MX2011011684A - Uses of immunoconjugates targeting cd138. - Google Patents

Abstract

Disclosed are methods and treatment regimes that include the administration of immunconjugates targeting CD138 to combat diseases. The immunoconjugate is either used as the sole active ingredient, as part of a treatment regime or as part of an anticancer combination.

Description

USES OF IMMUNOCONJUGATES DIRECTED TO CD138 FIELD OF THE INVENTION The present invention relates to methods and treatment regimens, in particular for human subjects, which include the administration of immunocytes that are designed to target cells expressing CD138. The present invention is also directed to anti-cancer combinations, to pharmaceutical compositions comprising the same, and to their uses in the treatment of cancers having target or CD138-expressing cells. The present invention in particular is directed to anti-cancer combinations that show synergy or other unexpected additive effects in the treatment treatment that involves less than all the components of the combination.

BACKGROUND CD138, which acts as a receptor for the extracellular matrix, is over-expressed in multiple myeloma (MM) cells and has been shown to influence the development and / or proliferation of MM cells. CD138 is also expressed in ovarian carcinoma cells, cervical cancer (Numa et al., 2002), endometrial cancer (Choi et al., 2007), kidney or kidney carcinoma, gallbladder carcinoma, vesicle-transitional cell carcinoma, Gastric cancer (Wiksten et al., 2008), prostate adenocarcinoma (Zell eger et al., 2003), mammary carcinoma (Loussouarn et al., 2008), non-small cell lung carcinoma (Shah et al., 2004), squamous cell lung carcinoma (Toyoshima et al., 2001), colon carcinoma cells and Hodgkin and non-Hodgkin lymphoma cells, colorectal carcinoma (Hashimoto et al, 2008), hepato-carcinoma (Li et al. ., 2005), chronic lymphocytic leukemia (CLL = Chronic Lymphocytic Leukemia), pancreatic carcinoma (Rabbit et al., 2000), and carcinoma of the head and neck (Anttonen et al., 1999) to name but a few.

The publications and other materials, including patents, herein used to illustrate the invention and in particular, to provide additional details regarding the practice, are incorporated by reference. For convenience, publications are referred to in the following text by author and date and / or are cited alphabetically by author in the attached bibliography.

Tassone et al. (2004) reported excellent binding of antibody B-B4 murine IgGl to the antigen CD138 expressed on the surface of M. Tassone cells also reported high cytotoxic activity of immunoconjugate B-B4-DM1, which comprises the maytansinoid DM1 as an effector molecule, against cells of multiple myeloma (see U.S. Patent Publication No. 20070183971).

Ikeda et al. (2008 and 2009) reported promising in vitro results and results in xenograft models with the BT062 immunoconjugate, which is based on B-B4.

While Tassone et al., And Ikeda et al., Represent contributions to providing effective treatment of MM and a composition of matter that can be employed in that treatment, there remain a number of needs to be satisfied in the art.

There remains in particular a need to provide convenient treatment regimens for diseases associated with CD138 expression, including plasma-proliferative disorders associated with CD138 expression, such as MM. Still more in particular remains a need for treatment regimens that ensure that toxicities to non-tumor cells, which also express CD138, are maintained at a clinically acceptable level, either by employing only certain tolerable amounts of immunoconjugate and / or by combining the immunoconjugate with cytotoxic agents known to be effective against the disorder in question. There is also a need for treatment regimens that reduce the need for medications that are used to relieve other symptoms of the disease.

The invention meets, in certain embodiments, one or more of these needs as well as other needs in the art that will be more apparent to the person skilled in the art once the following description is given.

COMPENDIUM OF THE INVENTION The invention fulfills one or more of the needs described above by the methods described herein for treating a disease associated with target cells expressing CD138.

The invention, in one embodiment, is directed to a method for treating a disease associated with target cells expressing CD138, comprising: administering to a subject, in particular a human subject, who requires it, an effective, but preferably tolerable, amount of an immunoconjugate comprising at least one directed agent, for example, an engineered engineered antibody that targets cells expressing CD138, and at least one effector molecule, wherein the targeted agent is functionally connected to the effector molecule to form the immunoconjugate.

Preferably at least a portion of the engineered engineered antibody preferably confers IgG4 isotype properties or alternatively any other immunoconjugate described herein.

In a further embodiment, the invention is an immunoconjugate for treating a disease associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally connected to the effector molecule to form the immunoconjugate, wherein the immunoconjugate is to be administered in an effective amount, and wherein the effective amount is a tolerable amount .

Additionally, in this embodiment, the invention is the use of an immunoconjugate for the manufacture of a medicament for treating a disease associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the immunoconjugate is to be administered in an effective amount, and wherein the effective amount is a tolerable amount.

The immunoconjugate is preferably administered to the subject in an amount of 5 mg / m2 to 200 mg / m2 or its pharmacokinetic equivalent.

A further preferred embodiment is a combined preparation of an immunoconjugate and an agent for treating adverse side effects, for simultaneous, separate or sequential use in the treatment of a disease associated with CD138-expressing target cells, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, and wherein the immunoconjugate is to be administered in an amount that is a pharmacokinetic equivalent of 5 mg / m2 to 200 mg / m2 of the immunoconjugate when administered alone.

Additionally, this additional preferred embodiment is the use of an immunoconjugate and an agent for treating adverse side effects for the manufacture of a combined preparation for simultaneous, separate or sequential use in the treatment of a disease associated with CD138-expressing target cells, wherein The immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate and wherein the immunoconjugate is to be administered in an amount that is a pharmacokinetic equivalent of 5 mg / m2 to 200 mg / m2 of the immunoconjugate when administered alone.

In particular, the immunoconjugate can be administered to the subject in an amount of 5 mg / m2 or 10 mg / m2 to less than 160 mg / m2, preferably 150 mg / m2, 140 mg / m2, 130 mg / m2 or 120 mg / m2.

The maximum concentration of the immunoconjugate in the plasma of the subject between 0 to 2 hours after the end of a first administration, can be less than 50%, preferably less than 40%, more preferably less than 30%, even more preferably less than 20%. %, or even less than 10% of a theoretical maximum concentration for the immunoconjugate.

The immunoconjugate can be administered at least four times and a maximum concentration of the immunoconjugate in the plasma of the subject between 0 to 2 hours after the end of each administration can be less than 55%, preferably less than 50%, more preferably less 40%, still more preferably less than 30%, less than 20% or even less than 10% of the theoretical maximum concentration for the immunoconjugate.

The maximum concentration can be less than 3 g / ml for 10 mg / m2; less than 8 g / ml for 20 mg / m2, less than 15 g / ml for 40 mg / m2, less than 25 g / ml for 80 mg / m2, less than 30 μg / ml for 120 mg / m2.

The maximum concentration of the immunoconjugate after a fourth application in the plasma of the subject between 0 to 2 hours after an end and a first administration may be less than 55%, preferably less than 50%, more preferably less than 40%, even more preferably less than 30%, less than 20% or even less than 10% of the theoretical maximum concentration for the immunoconjugate.

The maximum concentration can be less than 14 μg / ml for 20 mg / m2, less than 15 μg / ml for 40 mg / m2 or less than 25 pg / ml for 80 mg / m2. The immunoconjugate can be administered intravenously. The immunoconjugate can be administered intravenously in a single repeated dose and the maximum concentration of the immunoconjugate in the plasma of the subject between 0 to 2 hours after the end of either administration, can be less than 55%, less than 50% or less than 40%. % of the theoretical maximum concentration for the immunoconjugate. A stable disease can be maintained for at least 4, 5, 6, 7, 8, 9, 10 cycles of treatment (by at least 12, 15, 18, 21, 24, 27, 30 weeks). A state of at least stable disease can be maintained for 5, 6, 7, 8, 9 or 10 treatment cycles at 20 mg / m2 and optionally, the maximum concentration of the immunocontained in the subject's plasma 0 to 2 hours after a The end of any administration may be less than 55%, less than 50% or less than 40% of the theoretical maximum concentration for the immunoconjugate. In certain cases, a minor response can be observed after up to 8 treatment cycles.

The invention is also directed to a method for treating a disease associated with CD138 expressing target cells comprising administering to a subject, preferably a human subject,. that this treatment requires, an effective amount of an immunoconjugate comprising: at least one targeted agent that targets CD138 expressed on a cell surface, at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the immunoconjugate is administered in a dose, preferably a single repeated dose, not greater than about 10, 20, 30, 40, 80, 90, 100 or 120 mg / m2, an average daily dose of about 400 Ug / m2 to about 6 mg / m2, including about 500 pg / m2, about 1 mg / m2, about 2 mg / m2, about 3 mg / m2, about 4 mg / m2, and / or an average weekly dose of about 3 mg / m2 to about 40 mg / m2, including about 5 mg / m2, about 10 mg / m2, about 15 mg / m2, about 20 mg / m2, about 25 mg / m2, about 30 mg / m2 or ) approximately 35 mg / m2.

The methods referred to herein may allow stable disease maintenance for approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 210 or more days and / or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more treatment cycles each of approximately three weeks.

The invention is also directed to a method for treating a disease associated with CD138-expressing target cells comprising administering to a subject, preferably a human subject, requiring this treatment, an effective amount of an immunoconjugate comprising: ^ at least one targeting agent on CD138 expressed from cell surface, at least one effector molecule, wherein the targeted agent is functionally connected to the effector molecule to form the immunoconjugate, wherein CD138, in the subject is expressed in the target cells and in non-target cells, wherein the administration results in elimination or clearance in moderate or slow plasma, and wherein the non-target cells, in particular epithelial cells, are substantially not affected.

The effective amount that is administered may be less than 200 mg / m2 or less than a pharmacokinetic equivalent of 200 mg / m2 when administered in combination with an agent to treat adverse side effects and wherein the administration may result in a response of the subject , preferably after less than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5 hours.

The effective amount can be greater than 120 mg / m2.

The levels of CD138 expression in target and non-target cells (e.g., epithelial cells) may be comparable.

The effective amount can be administered as, for example, a single dose or a single or multiple dose repeated dose.

The effective amount can be administered in multiple doses, wherein the Cmax value after each administration is greater than 55% of the theoretical Cmax value.

The disease may be associated with bone pain and / or bone complications and administration of the immunoconjugate or an anti-cancer combination according to the present invention may reduce bone pain and / or bone complications, preferably to an acceptable level. The administration of medication to relieve bone pain and / or bone complications can be stopped or reduced from a base level commonly administered by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. The base level is the level that is generally recommended for the symptoms to be treated and can be evaluated from instructions for use that accompany the medication or that is known to the person with skill in the technique of administering pain medication.

For example, bisphosphonate, for example pamidronate, which is typically administered at 90 mg every four weeks or zoledroinic acid, which is typically administered at a dose of 4 mg once a month (Terpos et al., 2009) can be reduced by 10% , 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or at longer time intervals corresponding to this reduction) or can be eliminated.

The administration can also result in M or FLC protein levels of at least stable disease, a minor response or a partial response in the subject, preferably after a first administration.

The immunoconjugate can comprise an Antigen Binding Region (ABR = Antigen Binding Region) against CD138, and an additional antibody region, wherein at least part of the additional antibody region can be from a human antibody and can confer the properties of isotype IgG4.

The immunoconjugate may comprise nBT062 or a targeted antibody having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with nBT062 or it may correspond to BT062.

The subject can be a human subject.

The method can consist essentially of administering a pharmaceutical composition comprising the immunoconjugate and an acceptable pharmaceutical carrier, wherein an active ingredient of the composition can essentially consist of the immunoconjugate.

Any of the methods described here can result in a stable disease, a response, in particular a minor response, a partial response, a very good partial response, a severe complete response or a complete durable response by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 treatment cycles or more wherein the treatment cycles each comprise approximately 3 weeks with administration of the immunoconjugate on day 1 of each treatment cycle.

The invention is also directed to a method for treating a disease associated with CD138-expressing target cells, which comprises (i) identifying the disease as associated with target cells expressing CD138, such as multiple myeloma and not responding or responding poorly to treatment with one or more cytotoxic, immunomodulating agents such as lenalidomide and / or proteasome inhibitors such as bortezomib, and (ii) administering, preferably intravenously to the subject, an effective amount of an immunoconjugate as specified herein, at a dose of less than 200 mg / m2 when the immunoconjugate is administered alone or wherein the effective amount is the pharmacokinetic equivalent of 200 mg / m2 when administered with an agent to treat side effects, including potential side effects, wherein the subject does not respond, or responds poorly to treatment with one or more cytotoxic, immunomodulatory agents such as lenalidomide and / or proteasome inhibitors such as bortezomib, and wherein the disease is treated.

The invention is also directed to a method for treating a disease associated with CD138-expressing target cells, which comprises administer to a subject who requires this treatment and who exhibits high levels of sCD138, such as more than 50 ng / ml, more than 60 ng / ml, more than 70 ng / ml, more than 80 ng / ml, more than 100 ng / ml, more than 150 ng / ml, more than 200 ng / ml, more than 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 ng / ml, an amount effective of an immunoconjugate as specified herein, wherein an amount as low as 20 mg / m2 or as low as 40 mg / m2 is effective to result in a response such as a minor response. The response may result from the selective binding of the immunoconjugate. The subject may not respond or respond poorly to treatment with cytotoxic, immunomodulatory agents such as lenalidomide and / or proteasome inhibitors such as bortezomib.

The engineered engineered antibody may comprise an antigen binding region (ABR) against CD138, and an additional antibody region, wherein at least part of the additional antibody region is from a human antibody and confers the properties of isotype IgG4.

The disease can be multiple myeloma, in particular relapsed or refractory multiple myeloma.

The disease expressing CD138 in target cells can also be selected from the group consisting of renal cell carcinoma, endometrial cancer, cervical cancer, prostate adenocarcinoma, pancreatic carcinoma, gastric cancer, bladder cancer, mammary carcinoma, hepato-carcinoma, carcinoma colorectal, colon carcinoma, squamous cell carcinoma, lung cancer in particular squamous cell lung carcinoma, non-Hodgkin's lymphoma, thymus, uterus, urinary or ovarian carcinoma.

In preferred embodiments, the immunoconjugate targets homogenously in target or target cells expressing CD138.

In certain embodiments, the engineered engineered antibody of the present invention may (i) consists essentially of antigen binding region (ABR) against CD138 of a non-human antibody, or (ii) comprises an antigen binding region (ABR) against CD138, wherein the antigen binding region is a non-human antibody, and an additional antibody region, wherein at least part of the additional antibody region is from a human antibody.

The ABR may include: (a) CDR3 heavy chain variable region comprising amino acid residues 99 to 111 of SEQ ID NO: 1, and (b) CDR3 light chain variable region comprising amino acid residues 89 to 97 of SEQ ID NO: 2, respectively.

The ABR can also include: (a) heavy chain variable region CDR1 and CDR2 comprising amino acid residues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and / or (b) light chain variable region CDR1 and CDR2 comprising amino acid residues 24 to 34 and 50 to 56 of SEQ ID NO: 2, respectively.

The additional amino acid region may comprise: (a) amino acid residues 123 to 448 of SEQ ID NO: 1, and / or (b) amino acid residues 108 to 214 of SEQ ID NO: 2, respectively and its mutations that (i) maintain or reduce antibody-dependent cytotoxicity and / or cytotoxicity dependent on complement of engineered antibody and / or (ii) stabilize the engineered engineered antibody.

The antibody can comprise a light chain having at least about 70%, more preferably 80%, 85% or 90%, sequence identity with SEQ ID No: 2 and a heavy chain having at least about 70%, more preferably 80 %, 85% or 90%, sequence identity with SEQ ID No: 1, and comprises the antigen binding regions specified above. ' The effector molecule can be linked to the engineered antibody by a linker. The linker may comprise a disulfide bond. The effector molecule (e.g., DM4) can provide steric hindrance between the directed antibody and the effector molecule. The effector molecule can be at least one maytansinoid (e.g., DM1, DM3, or DM4) taxane or a CC1065, or its analogue.

The immunoconjugate can bind CD138 with a targeted variation of less than 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60% or 50%.

The immunoconjugate can, in certain embodiments of the methods described herein, comprise: a targeted agent that targets CD138, which comprises an isolated polypeptide comprising an amino acid sequence of an immunoglobulin heavy chain or a portion thereof, wherein the immunoglobulin heavy chain or its part has at least 70% sequence identity with SEQ ID NO: 1. A constant region of the immunoglobulin heavy chain of the part may be a constant region isotype IgG4.

The targeted agent of the immunoconjugate may comprise a light chain sequence having at least about 70% sequence identity with SEQ ID NO: 2. The targeted agent of the immunoconjugate may also comprise a heavy chain sequence having at least about 70% sequence identity with SEQ ID NO: l.

The present invention is also directed to a pharmaceutical composition comprising any of the immunoconjugates specified herein for the inhibition, retardation and / or prevention of tumor growth and / or dissemination of tumor cells, and one or more pharmaceutically acceptable excipients.

The pharmaceutical composition may include cytotoxic agents as specified herein.

The present invention is also directed to a kit comprising, in separate containers, the pharmaceutical composition in one or more dosage forms and in a separate container, instructions on how to administer the one or more dosage forms to a subject, in particular to a human subject that requires it, for example as a single repeated dose or another treatment regimen discussed herein.

In particular, in one aspect of the invention the administration of any of the immunoconjugates described herein is for a subject or cells of this subject, in particular a human subject, who benefits from said administration. The immunoconjugate can also be used for the manufacture of a medicament for the treatment of this disorder.

The invention further provides an immunoconjugate for treating a disease in a subject associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the subject does not respond, or responds poorly, to treatment with one or more cytotoxic agents including immunomodulators and / or proteasome inhibitors, and wherein the immunoconjugate is to be administered to the subject, preferably intravenously, in an amount of 5 mg / m2 to 200 mg / m2.

Additionally, the invention provides the use of an immunoconjugate for the manufacture of a medicament for the treatment of a disease in a subject associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the subject does not respond, or responds poorly, to treatment with one or more cytotoxic agents including immunomodulators and / or proteasome inhibitors, and wherein the immunoconjugate is to be administered to the subject, preferably intravenously, in an amount of 5 mg / m2 to 200 mg / m2.

The invention also provides a combined preparation of an immunoconjugate and an agent for treating adverse side effects, for simultaneous, separate or sequential use to treat a disease in a subject associated with CD138 expressing target cells, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the subject does not respond or responds poorly, to treatment with one or more cytotoxic agents including immunomodulators and / or proteasome inhibitors, and wherein the immunoconjugate is to be administered to the subject, preferably intravenously, in an equivalent pharmacokinetics of 5 mg / m2 to 200 mg / m2 of the immunoconjugate when administered alone.

In addition, the present invention provides the use of an immunoconjugate and an agent for treating adverse side effects for the manufacture of a combined preparation for simultaneous, separate or sequential use to treat a disease in a subject associated with CD138-expressing target cells, in where the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the subject does not respond, or responds poorly, to treatment with one or more cytotoxic agents including immunomodulators and one or more proteasome inhibitors, and wherein the immunoconjugate is to be administered to the subject, preferably intravenously, in an equivalent pharmacokinetics of 5 mg / m2 to 200 mg / m2 of the immunoconjugate when administered alone.

Additionally, the present invention provides an immunoconjugate for treating a disease in a patient associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the patient exhibits SCD138 levels in his plasma of more than 50 ng / ml, and wherein the immunoconjugate is preferably administered in an effective amount to provide at least a minor response.

The present invention also provides the use of an immunoconjugate for the manufacture of a medicament for the treatment of a disease associated with CD138-expressing target cells, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, where the patient exhibits sCD138 levels in his plasma, greater than 50 ng / ml, and wherein the immunoconjugate is preferably administered in an effective amount to provide at least a minor response.

In a preferred embodiment, the immunoconjugate is to be administered in an amount of at least 20 mg / m2 and more preferably at least 40 mg / m2.

In a preferred embodiment, the levels of sCD138 that the patient exhibits in the plasma is greater than 60 ng / ml, more than 70 ng / ml, more than 80 ng / ml, more than 100 ng / ml, more than 150 ng / ml. ml, more than 200 ng / ml, or more than 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 ng / ml.

The invention is also directed to an anti-cancer combination comprising at least one cytotoxic agent and at least one immunoconjugate comprising a targeting agent in cells expressing CD138, and at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein (a) the combination has a synergy ratio greater than 1, greater than 1.1, greater than 1.2, greater than 1.3, greater than 1.4, or (b) the combination has a synergistic ratio of about 1 and the effector molecule and the cytotoxic agent have interference modes of action, and wherein the anti-cancer combination is a pharmaceutical composition or a kit comprising at least the cytotoxic agent and the immunoconjugate of separate containers.

The cytotoxic agent can be a proteasome inhibitor, an immunomodulatory or anti-angiogenic agent, a DNA alkylating agent or a mixture of two more of these.

The cytotoxic agent can be bortezomib, thalidomide, lenalidomide, melphalan or a mixture of two or more of these.

The effector molecule and the cytotoxic agent of the anticancer combination can have modes of interference action and where these modes of action preferably involve microtubule inhibition or cell-cycle brake induction (melphalan, bortezomib and lenalidomide or thalidomide are cytotoxic agents that induce cell cycle brake). Alternatively, there may be modes of action without interference.

If the anticancer combination is part of the pharmaceutical composition, the pharmaceutical composition may comprise at least one acceptable pharmaceutical excipient.

The anti-cancer combination can also be part of a kit in which at least one cytotoxic agent and the immunoconjugate at least are stored in separate containers.

The invention is also directed to a method for treating a disease associated with CD138-expressing target cells, comprising: administering to a patient in need thereof, an effective amount of the anticancer combination mentioned herein or an anticancer combination comprising at least one cytotoxic agent and at least one immunoconjugate comprising a targeting agent in cells expressing CD138 and at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, and wherein the immunoconjugate overcomes a refractory phenotype of a patient against the cytotoxic agent.

The invention is also directed to a method for treating a disease associated with CD138-expressing target cells, comprising: administering to a patient in need thereof, an effective amount of an anti-cancer combination discussed herein and wherein the immunoconjugate overcomes a refractory phenotype.

The invention is also directed to a method for treating a proliferative disease without plasma associated with target cells expressing CD138, comprising: administering to an individual that requires it or to cells of the non-proliferative plasma disease, an effective amount of an immunoconjugate comprising: at least one targeting agent in cells expressing CD138, and at least one effector molecule, wherein the targeting agent is functionally linked to the effector molecule to form the immunoconjugate, wherein CD138 is in the subject, expressed in the target cells and in non-target cells at comparable levels or where CD138 in the subject is expressed in the target cells at levels lower than the non-target cells expressing CD138.

Non-target cells expressing CD138 may be epithelial cells.

The invention is also directed to a method for treating a non-proliferative plasma disease associated with CD138-expressing target cells, comprising: administering to a subject that requires it or to cells of the nonproliferative plasma disease, an effective amount of an immunoconjugate comprising at least one targeting agent in cells expressing CD138, and at least one effector molecule, wherein the targeting agent is functionally linked to the effector molecule to form the immunoconjugate, where the target cells of CD138 released by the disease over a period of 24 hours, 2, 3, 4, 5, 6 days.

The disease can be breast carcinoma.

The invention further provides a combined preparation of at least one cytotoxic agent and at least one immunoconjugate, for simultaneous, separate or sequential use to treat a disease in a subject associated with CD138-expressing target cells, wherein the immunoconjugate comprises: (i) a targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule at least to form the immunoconjugate, and where the subject has a refractory phenotype.

Additionally, the invention provides the use of at least one cytotoxic agent and at least one immunoconjugate for the manufacture of a combined preparation for simultaneous, separate or sequential use to treat a disease in a subject associated with CD138-expressing target cells, wherein the immunocon ugado comprises: (i) a targeting agent in cells expressing CD138 and (ii) at least one effector molecule wherein the targeted agent is functionally linked to the effector molecule at least to form the immunoconjugate, and wherein the subject has a refractory phenotype.

In a preferred embodiment, the combination of the cytotoxic agent at least and the immunoconjugate at least has a synergy ratio or ratio greater than 1, greater than 1.1, greater than 1.2, greater than 1.3 or greater than 1.4. Alternatively, the combination of the cytotoxic agent at least and the immunoconjugate at least has a ratio or synergistic ratio of about 1 to the effector molecule and the cytotoxic agent has overlapping or overlapping modes of action.

In a further aspect, the present invention provides an immunoconjugate for treating a non-proliferative plasma disease in a subject associated with CD138-expressing target cells, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (il) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, and wherein in the subject, CD138 is expressed in the target cells at comparable (equivalent) levels at or below the levels at which CD138 is expressed in non-target cells.

The invention also provides the use of an immunoconjugate for the manufacture of a medicament for treating a non-proliferative plasma disease in a subject associated with CD138-expressing target cells, wherein the immunoconjugate comprises: (i) at least one targeting agent in cells expressing CD138, and (ii) at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, and wherein in the subject, CD138 is expressed in the target cells at comparable levels (equivalent) to or less than levels at which CD138 is expressed in non-target cells.

The invention is also directed to a method for treating a non-proliferative plasma disease associated with CD138-expressing target cells, comprising: administering to an individual that requires it, or to cells of the nonproliferative plasma disease, an effective amount of an immunoconjugate comprising: at least one targeting agent in cells expressing CD138, and at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein the immunoconjugate induces remission of a solid tumor.

This remission can be a remission followed by a time interval that is free of new tumor growth (complete remission). This time interval may be greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, half a year or 1 year or more.

The solid tumor may be a pancreatic carcinoma or a mammary carcinoma.

The disease can be renal cell carcinoma, endometrial cancer, cervical cancer, prostate adenocarcinoma, pancreatic carcinoma, gastric cancer, bladder cancer, mammary carcinoma, hepato carcinoma, colorectal carcinoma, colon carcinoma, squamous cell carcinoma, lung cancer in particular squamous cell lung carcinoma, non-Hodgkin's lymphoma, thymus, uterine, urinary or ovarian carcinoma. i The solid tumor may be a mammary carcinoma, which is a negative estrogen receptor and / or a negative progesterone receptor.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 provides a schematic representation of nBT062 having effector molecules linked.

FIGURE 2 is a chemical representation of BT062.

FIGURE 3 shows the conversion of ansamitocin P-3 to maytansinol (stereochemistry is omitted for simplicity).

FIGURE 4 shows a representative synthesis scheme of DM4.

FIGURE 5 is a schematic representation of an antibody conjugation (nBT062 to DM4).

FIGURE 6 shows an analysis of the binding of nBT062-SPDB-DM4, nBTO 62-SPP-DM1, nBT062-SMCC-DM1 and antibody nBT062 to OPM-2 cells. Different concentrations of nBT062 and conjugates were delivered to the cells and average fluorescence was measured by FACS analysis.

FIGURES 7 (A) - (D) illustrate in vitro cytotoxicity of nBT062-D x conjugated to OLP-8 (CD138 +) and BJAB (CD138") cells.The cells were grown on flat bottom plates and incubated at the indicated concentrations of immunoconjugates for 5 days ST reagent is added for 3 more hours to estimate cell viability In (D), the cytotoxic activity of nBT062-SPDB-DM4 is analyzed in the presence or absence of blocking antibody (1 ju M nBT062).

FIGURE 8 shows tumor volumes for individual mice treated with (A) PBS, (B) nBT062 antibody, (C) free DM4 or (D) non-targeted conjugate huC242-DM4 with time (days) post-inoculation with cells from MOLP-8 tumor.

FIGURE 9 shows tumor volumes for individual mice treated with (A) PBS, (B) nBT062-SPDB-DM4, (C) B-B4-SPP-DM1 or (D) nBT062-SPP-DMl over time (days ) after inoculation with MOLP-8 tumor cells.

FIGURE 10 illustrates average tumor volume (+/- SD) of human MOLP-8 multiple myeloma xenografts in CB.17 SCID mice with time (days) post inoculation.

FIGURES 11A and B show anti-tumor activity of nBT062-DMx against CD138 + MOLP-8 tumor cells in a bulky MOLP-8 tumor model in SCID mice. Tumor volume is given as an average (+/- SD) for each group.

FIGURE 12 is a graph that reflects the anti-tumor efficacy of nBT062 containing DMx conjugated in the SCIDhu / INA-6 model to multiple myeloma cells in the human bone marrow environment. Soluble human IL-6 receptor produced by multiple myeloma cells (shuIL-6R) was used as an indicator for tumor loading. Triangle: nBT062-SPP-DMl, Square: nBT062-SPDB-DM4; Diamond: control vehicle.

FIGURE 13 shows extermination by nonspecific activation mediated by nBT062-SPDB-DM4, in vitro. OPM2 cells positive to CD138 and Namawla cells negative to CD138 were cultured with nBT062-SPDB-DM4 at different concentrations and cell viability was measured. OD450 values represent a measure for cell viability.

FIGURE 14 shows tumor growth curves in a mouse model of xenograft a single injection of BT062. The doses marked with an asterisk (*) are based on the molecular weight of bound DM4.

FIGURE 15 shows complete remission of a xenograft pancreatic carcinoma in mice treated with BT062 against a control.

FIGURE 16 shows the complete remission of a mammary carcinoma of xenograft in mice treated with BT062 against a control.

FIGURE 17 illustrates rapid plasma elimination for doses in the range of 40 mg / m2 to 120 mg / ra2, while higher doses as illustrated here by a dose of 160 mg / m2, showed a plasma clearance closer to the expected value.

FIGURE 18 compares the plasma profile of BT062 to monkeys treated at the same dose. The comparison clarifies that the rapid elimination of plasma at low doses can not be extrapolated from the available animal models and appears to be specific for humans.

FIGURE 19 shows the measured cmax values of BT062 compared to the theoretical Cmax values.

FIGURES 20 and 21 show that the Cmax values in general are similar over several treatment cycles.

FIGURE 22 clarifies that the rapid elimination of plasma can not be attributed to a buffering effect caused by soluble CD138.

FIGURE 23 illustrates a treatment diagram in human subjects with different doses of BT062 administered in the course of the indicated treatment cycles, wherein each treatment cycle lasted 21 days and the respective dose was administered on day 1 of each cycle.

Figure 24 shows the urine M protein level measured for a patient receiving 20 mg / m2 at three week intervals. Days 5 to 205 are shown.

Figure 25 shows the level of serum M protein measured by the patient receiving 40 mg / m2 at three-week intervals. Days 21 to 119 are shown.

The. Figure. 26 shows the level of kappa FLC measured for a patient receiving 160 mg / m2 at intervals of three weeks. Days 21 to 101 are shown.

Figure 27 shows the plasma concentration for BT062 in the cohort of 20 mg / m2 of patients.

Figure 28 shows the effects of combination therapy on mean tumor volume (TV = Tumor Volume) in a mouse xenograft model. The result shows the effects of the combination of BT062 and lenalidomide.

Figure 29 shows the effect of combination therapy on mean tumor volume (TV) in a mouse xenograft model. The result shows the effects of the combination of BT062 and VELCADE.

Figure 30 shows the effect of combination therapy on mean tumor volume (TV) in a mouse xenograft model. The result shows the effects of the combination of BT062 and melphalan.

DETAILED DESCRIPTION OF VARIOUS AND PREFERRED MODALITIES OF THE INVENTION The present invention relates to the administration of subjects, in particular human subjects (patients), who require it, of immunoconjugate comprising agents directed to CD138 described herein and the delivery of the effector molecule or molecules of the immunoconjugates to target sites and the release of one or more effector molecules on or at the target site, in particular cells, tissues and / or target organs. More particularly, the present invention relates to immunoconjugates comprising these agents directed to CD138 and potential effector molecules that bind to the targeted agents. The effector molecules can be activated by disruption and / or dissociation of the targeted agent portion of the immunoconjugate on or at the target site. The immunoconjugates can be administered alone or as part of an anticancer combination that includes a cytotoxic agent such as but not limited to, a proteasome inhibitor (e.g., bortezomib), anti-angiogenic agent / immunomodulatory agent (e.g., thalidomide or lenalidomide) , DNA alkylating agent (for example, melphalan) or corticosteroid (for example, dexamethasone), wherein the anti-cancer combination has synergy effects or unexpected additive effects in the treatment of cancer on the immunoconjugate used alone in monotherapy, the cytotoxic agent used only in monotherapy or both.

The immunoconjugates according to the present invention can be administered to a subject that requires treatment or to cells isolated from this subject that requires treatment. The effector molecule (s) can be released or detached from the immunoconjugate by dissociation / disruption in or within a cell or tissue and / or target organ.

In one example, the immunoconjugate BT062, which targets cells expressing CD138 by the nBT062 antibody and comprises DM4 as an effector molecule, was administered to a patient with refractory / relapse multiple myeloma four times in an amount of 80 mg / m2 as repeated single doses, where the duration of each treatment cycle was 21 days with the only dose / per cycle that is administered on day one of the cycle. In this example, the immunoconjugate was administered intravenously to the patient, so that it may be better concentrated in and / or within the tumor cells. Measurements of the plasma concentration of BT062 show that in an initial measurement phase (up to 2 hours after the end of administration) C max values for BT062 were significantly lower than the theoretical calculated value while no adverse side effects were observed, suggesting that BT062 it is concentrated on the tumor target instead of being randomly added to target and non-target CD138. A "damping effect" that results from sCD138 can be excluded (see Figure 20).

In another example, the BT062 immunoconjugate is administered to a patient with refractory / relapse multiple myeloma, ten times in an amount of 20 mg / m2 each as a single repeated dose, wherein the duration of each treatment cycle was 21 days with the only dose / per cycle that is administered on day one of the cycle. In this example, the immunoconjugate was administered intravenously to the patient in such a way that it could be better concentrated in and / or within the tumor cells. No additional means were provided to release the effector molecule from the immunoconjugate. Ten cycles of treatment were well tolerated and at least stable disease could be achieved for these treatment cycles.

In another example, the BT062 immunoconjugate was administered to a patient with multiple relapse myeloma four times in an amount of 160 mg / m2 as repeated single doses, wherein the duration of each treatment cycle was 21 days with the single dose / per cycle that is administered on day one of the cycle. In this example, the immunoconjugate was administered intravenously to the patient in such a way that it could be better concentrated in and / or within the tumor cells. At this concentration, plasma elimination was still lower than the theoretical Cmax, but not to the degree observed with lower doses. However, a sharp decrease in serum FLC level can be observed after a single treatment. A partial response could be observed after a 2nd, 3rd and 4th treatments.

In certain treatment regimens, the administration of medicament which alleviates pain and / or bone complications could be interrupted since the patient's pain diminished upon administration of the immunoconjugate.As a result, side effects associated with these medicaments (including bisphosphonates and another osteoporosis medication), such as osteronecrosis of the jaw.

In yet another example, the BT062 immunoconjugate is administered concomitantly to a patient with relapsing multiple myeloma, four times in an amount of 120 mg / m2 with a daily oral dose of 10 mg of the immunomodulatory agent lenalidomide as repeated single doses, in where the duration of each treatment cycle is 21 days with the only dose / per cycle that is administered on day one of the cycle. In this example, the immunoconjugate is administered intravenously to the patient, so that it may be better concentrated in and / or within the tumor cells.

In another example, the immunoconjugate BT062 is administered to a patient suffering from a pancreatic tumor, as repeated single doses, wherein the duration of each treatment cycle is 21 days with the only dose / per cycle that is administered on the day of the cycle . In this example, the immunoconjugate is administered intravenously to the patient in such a way that it can be better concentrated in and / or within the tumor cells.

CD138 or syndecan-1 (also described as SYNDl; SYNDECAN; SDC; SCD1; CD138 ANTIGEN, accession number SwissProt: P18827 human) is a membrane glycoprotein that was originally described as present in cells of epithelial origin, and subsequently was found in hematopoietic cells (Sanderson, 1989). CD138 has a long extracellular domain that binds to soluble molecules (eg, growth factors EGF, FGF, HGF) and insoluble molecules (eg, to the extracellular matrix components of collagen and fibronectin) through heparan sulfate chains ( Langford, 1998; Yang, 2007) and acts as a receptor for the extracellular matrix. CD138 also mediates cell-cell adhesion through heparin binding molecules expressed by adherent cells. It has been shown that CD138 has a role as a co-receptor for myeloma cell growth factors (Bisping, 2006). Differentiation studies of plasma cells show that CD138 should also be considered as an antigen of differentiation (Bataille, 2006).

In malignant hematopoiesis, CD138 is highly expressed in most MM cells, ovarian carcinoma, kidney carcinoma, gallbladder carcinoma, breast carcinoma, prostate cancer, lung cancer, colon carcinoma cells, and lymphoma cells of Hodgkin and non-Hodgkin's disease, chronic lymphocytic leukemia (CLL = Chronic Lymphocytic Leukemia) (Horvathova, 1995), acute lymphoblastic leukemia (ALL = Acute Lymphoblastic Leukemia), acute myeloblastic leukemia (AML = Acute Myeloblastic Leukemia) (Seftalioglu, 2003 (a ); Seftalioglu, 2003 (b)), solid tissue sarcomas, colon carcinomas as well as other hematological malignancies and solid tumors expressing CD138 (Carbone et al., 1999; Sebestyen et al., 1999; Han et al., 2004 Charnaux et al., 2004; O'Connell et al., 2004; Orosz and Kopper, 2001). The expression of CD138 is also associated with different types of gastrointestinal malignancies (Conejo et al., 2000).

As illustrated in Table 1, a number of tumorigenic cell lines exist associated with expression / overexpression of CD138.

Sensi Origin LineBili CD138 Cell Expression dad IC50 RFI * Recep- (nM) tores / - cell NCI-H929 MM 0.38 502 788,752 PC-3 Cancer 0.79 541 195, 671 prostate U266 MM 1.59 617 782, 987 MOLP-2 MM 1.78 425 161, 064 SK-BR-3 Carcinoma of 2.72 485 444, 350 breast LNCaP Cancer of 7.39 179 23, 388 prostate CAPAN-2 Carcinoma of 15.51 328 n. d. pancreas PANC-1 Carcinoma of 36.38 34 18,085 pancreas T47D Carcinoma 89.28 217 42.264 breast Jurkat Lymphoma of 39.00 n. 0 T cells Table 1: CD138 expression in different cell lines. In the context of MM it was shown that sensitivity to BT062 correlates with higher expression of CD138 (relative fluorescence index, RFI = Relative Fluorescence Index).

The sensitivity observed, for example, of breast carcinoma cell lines and pancreatic carcinoma cell lines were substantially lower than those of MM cell lines. However, as described in the experimental section in mouse xenograft models using cells from patients with breast cancer and pancreatic cancer, not only were comparable results obtained, but significantly better than comparable xenograft models for MM. In both cases, complete remission could eventually be obtained, while comparable MM models showed a marked delay in tumor growth, but not complete remission.

While in pancreatic cancer there seems to be no difference in expression of syndecan-1 mRNA, between early or early and advanced tumors, in mammary carcinoma, it was reported that CD138 can be lost over time as reflected by weak or lacking IHC staining. Loss of CD138 expression has been reported and often correlated with a change in expression, ie de novo expression in the surrounding stroma (Loussouarn, 2008). As a result, fewer targets for agents targeting CD138 can be expected over time.

Other cancers that have been shown to be positive for CD138 expression are many adenocarcinomas of ovaries, bladder transition cell carcinomas, clear cell carcinomas of the kidney, squamous cell lung carcinomas; and uterine cancers (see, for example, Davies et al., 2004, Barbareschi et al., 2003, Mennerich et al., 2004, Anttonen et al., 2001, Wijdenes, 2002).

The treatment of active multiple myeloma (symptomatic) and related plasmaproliferative disorders will serve as an example of diseases that can be treated by immunoconjugates of the present invention.

Plasma-proliferative disorders as used herein, mean hematological and / or plasma cell disorders such as MGUS, SMM, Active MM (symptomatic), Waldenstrom's macroglobulinemia, solitary plasmacytoma, systemic AL amyloidosis and POEMS syndrome.

Multiple myeloma (MM) refers to a malignant proliferation of plasma cells that typically originates in the bone marrow, primarily involves the skeleton of a patient and presents clinical features that are attributed to particular sites of involvement and abnormalities in protein formation of plasma. The condition is usually characterized by numerous nodular accumulations or diffuse foci of abnormal or malignant plasma cells in the marrow of various bones (especially the skull), causing palpable swelling of the bones, and occasionally in extraskeletal sites. Upon radiological examination, bone lesions may have a characteristic "punctured" appearance. Cells involved in myeloma typically produce abnormal proteins and / or abnormal protein levels in serum and urine. The disease typically develops monoclonal gammopathy of undetermined significance (GUS = Monoclonal Gammopathy of Undetermined Significance) to indolent multiple myeloma (SMM = Multiple Smoldering Myeloma) to active multiple myeloma (MM = Active Multiple Myeloma). Symptoms of these conditions vary, but may include hypercalcemia, kidney failure, fatigue, anemia, bone pain, spontaneous fractures, increased frequency or duration of infection, or abnormal color or odor of urine. When the present invention relates to Multiple Myeloma it refers to (MGUS), indolent multiple myeloma (SMM) and active multiple myeloma (MM) as well as other malignant proliferation of plasma cells that can eventually develop into active MM.

MGUS, a clinically benign precursor condition of MM is more common than MM, occurring in 1% of the population over 50 years of age and 3% of those over 70 years of age (Greipp and Lust, 1995). It is important to distinguish patients with MGUS from those with MM, since MGUS patients can be observed safely without resorting to therapy.

However, during long-term follow-up, of 241 patients with MGUS, 59 patients (24.5%) continue to develop MM or a related disorder (See Kyle et al., 1993).

The term "gamopathy" refers to a primary disturbance in immunoglobulin synthesis of a patient.

Monoclonal gammopathy refers to any of a group of disorders that are typically associated with the proliferation of a single clone of plasma or lymphoid cells (normally visible in serum protein electrophoresis (SPEP = Serum Protein ElectroPhoresis) as a single peak) and characterized by the presence of monoclonal immunoglobulin in the serum or urine of a patient.

Indolent MM (SMM = Smoldering M) has been reported to precede the onset of symptomatic multiple myeloma in the elderly. Indolent multiple myeloma is often considered as an advanced phase of MGUS; even at the time of progression, multiple myeloma evolved from indolent multiple myeloma usually lacks osteolytic lesions or other cardinal features of symptomatic multiple myeloma.

Clinical symptoms of MM include anemia, hypercalcemia, renal failure and lytic bone lesions. Distinctions in the course and severity of the disease as monoclonal gammopathy of undetermined significance (MGUS) is developed to indolent multiple myeloma (SMM) to multiple myeloma (MM) are given in Table 2 below. The table also summarizes methods of detection, diagnosis and supervision of these conditions. These symptoms and techniques are familiar to those with skill in the specialty.

TABLE 2 Comparison of Characteristics Clinics of MM, SMM, or MGUS Features MM SMM MGUS Plasma cells of marrow > = 10% > = 10% < 10% M protein of > = 3 serum > = 3 g / dL g / dL < 3 g / dL < 1 g / 24 < 1 g / 24 Bence-Jones > = 1 g / 24 h h Urine protein Yes Yes Yes Usually Au¬ Anemia present be sente Hypercalcemia, It may be absent present absent Bone injuries Usually lytic present absent Absent MM = myeloma multiple SMM = myeloma multiple indolent MGUS = monoclonal gamopathy of indeterminate significance Staging stages by clinical characteristics and severity of multiple myeloma Stage of advance of illness Stage I (MM active) Relatively few cancer cells are spread throughout the body. The number of red blood cells and the amount of calcium in the blood are normal.

No tumors (plasmacytomas) are found in the bone. The amount of M protein in the blood or urine is very low. There may be no symptoms of illness.

Stage II (MM active) A moderate number of cancer cells has disseminated through body Stage III (MM active) A relatively large number of cancer cells have spread throughout the body.

There may be one or more of the following: A decrease in the number of red blood cells, causing anemia.

The amount of calcium in the blood is very high, because the bones get damaged.

More than three bone tumors (plasmacytomas) are found.

High levels of M protein are found in the blood or urine.

Characteristics MM clinics 1 Hyperealeemia Insufficiency renal Anemia Protein monoclonal: SPEP (electrophoresis of protein serum) SPIEP (immunoelectrophoresis of whey protein) Immunoelectrophoresis of protein in urine (protein Bence - Jones protein) Diagnosis of MM > 10% of plasma cells in marrow or aggregates in biopsy or plasmacytoma Protein monoclonal: Protein M from serum > 3 g / dl or Protein M in urine Active multiple leiomyeloma (MM) is typically recognized clinically by the proliferation of malignant plasma cells in the bone marrow of a patient. These neoplastic plasma cells produce immunoglobulins and evolve from B lymphocytes. Immunoglobulins that are produced by plasma cells can be detected in the blood serum and / or urine of a patient by electrophoresis test.

As indicated in Table 2, measurement of serum M protein is an important tool for estimating MM in different stages.

"Protein M" refers to a monoclonal protein that is typically visualized as a narrow band in electrophoretic gel, or an abnormal arc in immunoelectrophoresis. It represents a proliferation of homogeneous immunoglobulin produced by clone cells originating from a single common cell, for example a monoclonal immunoglobulin characterized by a heavy chain of a single class and sub-class, and light chain of a single type (also referred to as peak M and more broadly as paraprotein).

"Serum protein electrophoresis" (SPE or SPEP) and "electrophoresis immunofixation" (IFE) can detect monoclonal immunoglobulin, which occurs in several proliferative disorders of plasma cells including multiple myeloma (MM). Across the population, up to 61% of these findings are not associated with clinical symptoms, allowing a diagnosis of monogamopathy of undetermined significance (MGUS). SPE and IFE, however, do not detect all monoclonal immunoglobulins, particularly when only light chains are secreted.

Those "free light chain molecules" (FLCs = "Free Light Chain Molecules") include light chains? Y ?. Plasma cells produce one of the five types of heavy chains along with any of the molecules? or? This normally is approximately 40% excess production of free light chain on heavy chain synthesis. Plasma cells secrete free light chains (FLC, kappa or lambda) in addition to intact immunoglobulin molecules, and at serum light chain levels - they are determined by the relative proportions of synthesis (? >;?) and renal excression (? &?). In the presence of a monoclonal immunoglobulin, the proportions?:? they may already be higher or lower than the normal range, depending on the type of FLC involved. The serum half-life of FLCs is 2-6 hours, compared with 5 days for IgA, 6 days for IgM and 21 days for IgG. In this way, the measurement of serum CRF levels allows for a much faster evaluation of tumor response to therapy than the measurement of intact immunoglobulin. Likewise, serum FLC measurements allow early detection of relapse.

Nonproliferative plasma diseases are also associated with CD138 expression.

Pancreatic carcinoma The majority of cases comprise exocrine type. Most of these exocrine cancers represent ductal adenocarcinoma (more rapid subtypes include cystic tumors, acinar cell tumors, and sarcoma). Endocrine cancer of the pancreas represents a tumor that produces hormone.

Carcinoma in situ refers to the early stage of cancer, when it is confined to the cell layer where it started. In breast cancer, in situ means that cancer cells remain confined to ducts (ductal carcinoma in situ) or lobes (lobular carcinoma in situ). They have not grown in tissues deeper in the breast or disseminated to other organs in the body, and are sometimes referred to as non-invasive or pre-invasive breast cancers.

Invasive carcinoma (infiltrating).

The exocrine cells and endocrine cells of the pancreas form completely different types of tumors.

Exocrine tumors These are by far the most common type of pancreatic cancer and most pancreatic exocrine tumors are malignant. Approximately 95% of cancers of the exocrine pancreas are adenocarcinomas (an adenocarcinoma is a cancer that begins in gland cells). These cancers usually start in the ducts of the pancreas, but sometimes develop from cells that make up the pancreatic enzymes (acinar cell carcinomas).

Less common types of ductal cancers of the exocrine pancreas include adenosquamous carcinomas, squamous cell carcinomas, and giant cell carcinomas.

Endocrine tumors Tumors of endocrine pancreas are not common. As a group, they are known as pancreatic neuroendocrine tumors (NETs = neuroendocrine tumors), or sometimes i as islet cell tumors. There are several subtypes of islet cell tumors. Each one is named according to the type of hormone-producing cell in which it starts: The main system used to describe the stages of exocrine pancreatic cancers is the TNM system of the American Joint Cancer Committee (AJCC = American Joint Committee on Cancer (AJCC) as provided by the American Cancer Society (ACS = American Cancer Society). The TNM system for classification contains 3 key parts of information: T describes the size of the primary tumor (s), measured in centimeters (cm), and whether the cancer has spread within the pancreas or to nearby organs. Distinctions are made between TX, TO, TI, T2, T3 and T4, where a higher number indicates the progression of the disease.

N describes dissemination to nearby lymph nodes (regional). The N categories include NX, NO and NI.

M indicates whether the cancer has metastasized (spread) to other organs of the body. (The most common sites of disseminated pancreatic cancer are the liver, lungs, and peritoneum - the space around the digestive organs. M categories include: MX, MO and MI.

After the categories T, N and M have been determined, this information is combined to assign a stage, a process called grouping by phases or stages.

Stage 0 (Tis, NO, MO): The tumor is confined to the upper layers of pancreatic duct cells and has not invaded deeper tissues. It has not spread outside the pancreas. These tumors are sometimes referred to as pancreatic carcinoma in situ or pancreatic intraepithelial neoplasia III (Panln III).

Stage IA (TI, NO, MO): The tumor is confined to the pancreas and is less than 2 cm in size. It has not spread to nearby lymph nodes or distant sites.

Stage IB (T2, NO, MO): The tumor is confined to the pancreas and is larger than 2 cm in size. It has not spread to nearby lymph nodes or distant sites.

Stage IIA (T3, NO, MO): The tumor grows outside the pancreas but not in large blood vessels. It has not spread to nearby lymph nodes or distant sites.

Stage IIB (Tl-3, NI, MO): The tumor is already confined to the pancreas or growing outside the pancreas but not in large blood vessels or nearby large nerves. It has spread to nearby lymph nodes but not to distant sites.

Stage III (T4, Any N, MO): The tumor grows outside the pancreas in large blood vessels or nearby large nerves. It may or may not have spread to nearby lymph nodes. It has not spread to distant sites.

Stage IV (Any T, Any N, MI): The cancer has spread to distant sites.

Although formally not part of the TNM system, other factors are also important in determining prognosis (outlook). The degree of cancer (how abnormal the cells would be under a microscope), is sometimes cited on a scale of Gl to G4, with Gl cancers that look like more normal-like cells and that) They have the best perspective.

For patients who have undergone surgery, another important factor is the extent of the resection - whether or not the entire tumor was removed. This is sometimes cited on the scale of RO (where all visible and microscopic tumors were removed) to R2 (where some visible tumor could not be removed).

From a practical point of view, how far the cancer has spread often can not be determined accurately without surgery. This is why doctors often use a simpler classification system, which divides cancers into groups based on whether or not they are likely to be surgically removed. These groups are called resectable, or they can be operated, locally advanced (not resectable), and metastatic. These terms can be used to describe both exocrine and endocrine pancreatic cancers.

Resectable: If the cancer is only in the pancreas (or has spread just beyond it) and the surgeon can remove the tumor, it is called resectable.

Advanced locally (not resectable): If the cancer has not yet spread to distant organs but can not yet be completely removed with surgery, it is called locally advanced. Often the reason why the cancer can not be removed is because too much of it is present in nearby blood vessels.

Metastatic: when the cancer has spread to distant organs, it is called metastatic. Surgery can still be done, but the goal would be to alleviate the symptoms not cure the cancer.

Pancreatic neuroendocrine cancers are not classified as cancers of the exocrine pancreas. On the contrary, the statistics are broken down into different stages: localized (only in the pancreas), regional (disseminated to nearby tissues or lymph nodes), and distant (spread to distant sites, such as the liver).

Bladder tumors are grouped by the way in which cancer cells are seen under a microscope.

Transitional cell carcinoma (also called urothelial carcinoma) is by far the most common type of bladder cancer. Among this group are also subtypes. They are named depending on the shape of the cells and if they tend to spread and invade other organs. (If they are likely to grow deeper inside the bladder wall they are called invasive, if they are not likely to be invasive.) These tumors are divided into degrees based on how the cells look under a microscope. If the cells look more like normal cells, the cancer is called a low-grade cancer. When the cells look very abnormal, the cancer is high grade. Low-grade cancers tend to grow more slowly and have a better outcome than higher-grade cancers.

Also included in the definition, squamous cell carcinoma (not common, usually invasive); adenocarcinoma (not common, almost all are invasive); small cells (rare). Other rare bladder cancers are also included in this definition.

Bladder cancer is also classified: Stage Oa (Ta, NO, MO): Cancer is a non-invasive papillary carcinoma. It has growth toward the hollow center of the bladder but has no growth to the muscle or connective tissue of the bladder wall.

It does not spread to lymph nodes or distant sites.

Stage OIS (Tis, NO, MO): The cancer is flat, noninvasive carcinoma, also known as flat carcinoma in situ (CIS = carcinoma in situ). Cancer grows in the lining of the bladder only. Neither has inward growth to the hollow part of the bladder nor has it invaded the muscle or connective tissue of the bladder wall. It has not spread to lymph nodes or distant sites. Stage I (IT, NO, MO): The cancer has grown in the connective tissue layer under the bladder lining without growth in the thick layer of muscle in the bladder wall. The cancer has not spread to lymph nodes or distant sites.

Stage II (T2, NO, MO): The cancer has grown inside the thick muscle layer of the bladder wall but has not completely passed through the muscle to reach the layer of fatty tissue that surrounds the bladder. The cancer has not spread to lymph nodes or distant sites.

Stage III (T3 or T4a, NO, MO): The cancer has grown completely through the bladder within the layer of fatty tissue that surrounds the bladder (T3). It may have spread to the prostate, uterus or vajina (T4a). It does not grow inside the pelvic or abdominal wall. The cancer has not spread to lymph nodes or distant sites.

Stage IV (T4b, NO, MO) or (any T, N 1 to 3, MO) or (any T, any N, MI): Cancer has spread through the wall of the bladder to the pelvic or abdominal wall (T4b) and / or has spread to lymph nodes (Nl-3) and / or distant sites such as bones, liver or lungs (Mi ). ( Types of gallbladder carcinoma More than 9 out of 10 gallbladder cancers are adenocarcinoma. An adenocarcinoma is a cancer that begins in cells with gland-like properties that line many internal and external surfaces of the body (including the inside of the digestive system).

A type of gallbladder adenocarcinoma that deserves special attention is called papillary adenocarcinoma or just papillary cancer. These are gallbladder cancers whose cells are arranged in finger-like projections when viewed under a microscope. In general, papillary cancers are not likely to grow inside the liver or lymph nodes. close. They tend to have a better prognosis (outlook) than most other types of gallbladder adenocarcinomas. Approximately 6% of all gallbladder cancers are papillary adenocarcinomas.

There are other types of cancer that can develop in the gallbladder, such as adenosquamous carcinomas, squamous cell carcinomas, and small cell carcinomas, but these are not common.

The following stages of gallbladder carcinomas are distinguished based on the TNM system of AJCC: Stage 0: Tis, NO, M0: This is a small cancer only in the epithelial layer of the gallbladder. It has not spread outside the gallbladder.

Stage IA: TI (a or b), NO, MO: The tumor grows in the lamina propria (Aunt) or the muscular layer (Tlb). It has not spread outside the gallbladder.

Stage IB: T2, NO, MO: The tumor grows within the perimuscular fibrous tissue. It has not spread outside the gallbladder.

Stage IIA: T3, NO, MO: The tumor extends through the serous layer and / or directly grows within the liver and / or one or more nearby structures. It has not spread to lymph nodes or distant tissues or organs of the gallbladder.

Stage IIB: Ti to T3, NI, MO: In addition to any growth in the gallbladder, the tumor has spread to nearby lymph nodes (NI). It has not spread to distant tissues or organs of the gallbladder.

Stage III: T4, any N, MO: The tumor invades the main blood vessels that direct within the liver or has reached more than one nearby organ other than the liver. It may or may not have spread to the lymph nodes. It has not spread to tissues or organs away from the gallbladder.

Stage IV: Any T, any N, MI: The tumor has spread to distant tissues or organs of the gallbladder.

Mammary carcinoma An adenocarcinoma generally refers to a type of carcinoma that starts in glandular tissue (tissue that produces and secretes a substance). In the context of breast cancer, the ducts and lobes of the breast are glandular tissue, so that cancers that start in these areas are often called adenocarcinomas. There are several types of breast cancer, although some of them are quite rare. In some cases, a single breast tumor may have a combination of these types or have a mixture of invasive and in situ cancer.

Institut ductal carcinoma (DCIS, also known as intra ductal carcinoma) is the most common type of non-invasive breast cancer.

Invasive ductal carcinoma (or infiltrative) (IDC = invasive ductal carcinoma) is the most common type of breast cancer. Invasive (or infiltrative) ductal carcinoma (IDC) begins in a milk passage (duct) of the breast, breaks the wall of the duct and grows into the fatty tissue of the breast. At this point it may be able to spread (metastasize) to other parts of the body through the lymphatic system and the bloodstream. Approximately 8 to 10 invasive breast cancers are infiltrating ductal carcinomas.

IDC patients reveal expression of CD138 (Loussouarn et al., 2008).

Triple-negative breast cancer describes breast cancers (usually invasive ductal carcinomas) whose cells lack estrogen receptors and progesterone receptors, and do not have an excess of the HER2 protein on their surfaces. Triple negative breast cancers tend to grow and spread more quickly than most other types of breast cancer. Because tumor cells lack these certain receptors, neither hormone therapy nor drugs that target HER2 are effective against these cancers (although chemotherapy can still be useful, if required).

Some other breast cancers. that fall under the term "mammary carcinoma" are inflammatory breast cancer, marrow carcinoma, metaplastic carcinoma, mucinous carcinoma, tubular carcinoma, papillary carcinoma, adenoid cystic carcinoma (adenocystic carcinoma), phyllodes tumor.

Surgery, radiation or chemotherapy are standard cancer therapies. Hormone therapy is used occasionally. Hormone therapy is a form of systemic therapy. It is most often used as adjuvant therapy to help reduce the risk of cancer recurrence after surgery, although it can be used as a neoadjuvant treatment equally. It is also used to treat cancer that has come back after treatment or has spread. Estrogen promotes the growth of approximately 2 of 3 breast cancers - those that contain estrogen receptors (ER-positive cancers) and / or progesterone receptors (PR-positive cancers). Because of this, several approaches to block the effect of estrogen or reduce estrogen levels, are used to treat ER-positive and PR-positive breast cancers. However, hormone therapy is ineffective for patients who lack ERs or PRs.

Carcinoma mammary also follows this system of classifications: Stage 0: Atypical cells have not spread outside the ducts or lobules, the milk-producing organs, within the surrounding breast tissue. Referred to as carcinoma in situ, it is classified into two types: "In Situ Ductal Carcinoma" (DCIS), which is a very early cancer that is highly treatable and is of survival and "Lobular Carcinoma In Situ" (LCIS), which is not a cancer but an indicator that identifies a woman who has an increased risk of developing breast cancer.

Stage I: The cancer is not larger than two centimeters (approximately one inch) and has not spread to surrounding lymph nodes or outside the breast.

Stage II: This stage is divided into two categories according to the size of the tumor and whether or not it has spread to the lymph nodes: Breast cancer Stage II A - the tumor is less than two centimeters long and has spread to up to three auxiliary axillary lymph nodes. 0, the tumor has grown more than two centimeters, but not more than five centimeters and has not spread to the surrounding lymph nodes.

Breast cancer Stage II B - the tumor has grown between two and five centimeters and has spread to three auxiliary axillary lymph nodes. Or, the tumor is larger than five centimeters, but it has not spread to the surrounding lymph nodes.

Stage III: This stage is also divided into two categories: Breast cancer Stage III A - the tumor is larger than two centimeters but smaller than five centimeters and has spread to up to nine auxiliary axillary lymph nodes.

Breast cancer Stage III B - the cancer has spread to tissues near the breast including the skin, chest wall, ribs, muscles or lymph nodes in the chest wall or above the clavicle.

Stage IV: Here, the cancer has spread to other organs or tissues, such as the liver, lungs, brain, skeletal system or lymph nodes near the clavicle.

Lung cancer There are 4 types of neuroendocrine lung tumors, ie large cell neuroendocrine carcinoma, atypical carcinoid tumor, typical carcinoid tumor, and small cell lung cancer. Carcinoid tumors are tumors that start from cells of the diffuse neuroendocrine system. Typical and atypical carcinoid tumors look different under the microscope. Typical carcinoids grow slowly and only rarely spread beyond the lungs and approximately 9 out of 10 lung carcinoids are typical carcinoids.

For treatment purposes, two main types of lung cancer, which are treated very differently, are differentiated, namely small cell lung cancer (SCLC = Small Cell Lung Cancer) and non-small cell lung cancer (NSCLC = Non-Small). Cell Lung Cancer). If the cancer has characteristics of both types, it is called large cell / mixed small cell cancer.

Approximately 10% to 15% of all lung cancers are the same cell type. Other names for SCLC are microcytic carcinoma and undifferentiated small cell carcinoma.

This cancer often starts in the bronchi near the center of the chest. Although cancer cells are small, they can divide rapidly, form large tumors, and spread to lymph nodes and other organs throughout the body. Surgery is rarely an option and is never the only treatment provided. Treatment includes cytotoxic agents, such as drugs to determine broad / extensive disease.

There are 3 sub-types of NSCLC, ie squamous cell carcinoma; adenocarcinoma; Large cell carcinoma (undifferentiated).

Classification of non-small cell lung cancer The system used to classify non-small cell lung cancer is the system of the American set for cancer (AJCC = American Joint Committee on Cancer). The classifications are described using Roman numerals from 0 to IV (0 to 4). Some classifications are further divided into A and B. As a rule, the smaller the number the less cancer has spread. Higher number, such as stage IV (4), means more advanced cancer.

A respective classification system, including Classes I to IV, was also developed for squamous cell carcinoma (head and neck cancer).

Class I cancers are localized and usually curable cancers, class II and III, typically are locally advanced and / or have spread to local lymph nodes, and Class IV cancers are usually metastatic (which have spread to distant parts of the body). body) and are generally considered inoperable.

The treatment in the context of the present invention includes preventing or slowing progression, stabilizing the disease state, remitting the disease 0 improve one or more symptoms of a disorder associated with cells expressing CD-138. Treatment in this way includes avoiding or stopping the increase in severity or the remission of the disorder. In the case of MM, generally only patients with active MM class II or III receive primary therapy (Class I patients with SMM are initially only seen at intervals of 3 to 6 months), a treatment according to the present invention does not only include treatment for example of any active class of MM, but also includes the treatment of forms of disease states that precede the traditionally treated disease state. The particular treatment also includes avoiding the progression from one disease state to the next: in the case of MM, this would for example be the way of progression from MGUS to SMM or from SMM to class 1 MM active or another MM class. In the case of exocrine pancreatic cancers, for example a progression from Class I to Class II, including any worsening as reflected by the categories established by AJCC within the classes, for example from IA to IB. However, the term also includes maintaining the status quo, such as maintaining stable disease and, as discussed below, producing certain responses in the treated patient. A patient is also "successfully" treated if the patient shows observable and / or measurable reduction in or in the absence of inter alia, one or more of the following: reduction in the number of cancer cells or absence of cancer cells; reduction in tumor size; inhibition (i.e., slowing to a certain extent and preferably stopping) the infiltration of cancer cells into peripheral organs including the spread of cancer within the soft tissue and bone; inhibition (that is, slowing to a certain extent preferably stopping) tumor metastasis; inhibition, to some extent of tumor growth; and / or relief to some extent from one or more of the symptoms associated with the specific cancer; reduce morbidity and mortality, and improve the quality of aspects of life. In general, an effect of a certain treatment on a patient's disease state can be monitored, in the case of M, by measuring the levels of M protein in the patient's serum and / or urine and / or the FLC levels in the patient. serum and / or urine of the patient. In the case of other disorders associated with cells expressing CD-138, other parameters are measured to estimate the effect of a treatment according to the present invention. CRP is a non-specific inflammation parameter for clinical monitoring of cancer. To name but a few, for pancreatic cancer, relevant parameters that can be measured are CA 19-9 (carbohydrate antigen 19.9, a tumor marker often elevated in pancreatic cancer), bilirubin, or C-reactive protein. In addition, image formation such as sonography, CT, MRT is employed. In head and neck cancer, biomarkers that depend on the type of tumor are used (for example, SCC for squamous cell carcinoma, NSE: for erkel cell, CEA); in breast carcinoma,. the expression of CA 15-3Her2 and the expression of Cadherin can be used as markers, while the treatment supervised by serum markers such as neuron-specific enolase (NSE = Neuron Specific Enolase).

Bladder tumor antigen (BTA = Bladder Tumor Antigen) and NMP22 tests can be used together with cytoscopy (using a thin lighted tube to observe the bladder) to diagnose the condition in symptomatic subjects. These tests are also used to follow up on some patients after treatment, although cytoscopy and urine cytology (using a microscope to look for cancer cells in the urine) are still recommended as standard tests for diagnosis and follow-up. Tests ??? and NMP22 are often used between cytoscopies. Normal values may allow cytoscopy to be performed less often. However, these tests can not replace cytology and urine cytoscopy.

For advanced bladder cancer, some of the markers used for other cancers such as CEA, CA 125, CA 19-9, and TPA may be elevated and may be used to monitor patients during and after treatment. For lung cancer, there are no established markers, they can be elevated, CEA and NSE.

Tumor cells, such as myeloma cells or mammary carcinoma cells are known to shed CD138. The loss of CD138 from surfaces is correlated with a poor prognosis in myeloma. High levels of soluble CD138 have also been detected in other oncological indications such as cancer of the neck and neck or lung (Anttonen et al., 1999). The loss of surface Syndecan-1 correlates with the epithelial mesenchymal transition (EMT = Epithelial Mesenchymal Transition) this process describes the transformation of a malignant cell into a poorly or less differentiated cell associated with invasiveness and metastatic stage. This, for example, is reported for metastatic breast cancer (Loussouarn et al., 2008).

An effective amount of an agent, in particular an immunoconjugate or a pharmaceutical composition comprising an immunoconjugate according to the present invention, refers to an amount required to "treat" a disease or disorder in a subject, in particular a human subject ( patient). In the case of cancer such as MM, the effective amount of the agent can reduce the number of cancer cells; reduce the size of tumor; inhibit (i.e., slow to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie stop to a certain extent and preferably stop) tumor metastasis; inhibit to some extent, tumor growth; and / or alleviate to some extent one. or more of the symptoms associated with cancer. See the definition here of "treatment".

"A pharmacokinetic equivalent" of, for example, 200 mg / m2 refers to the amount of immunoconjugate that results in equal pharmacokinetics observed at a dose of 200 mg / m2 when the immunoconjugate is administered in combination, including co-administering with an agent for current treatment including potential adverse side effects primarily in non-target cells that also express CD138. Those equivalents can be something less than 200 or something more than 200, depending on the other agent. For example, effective amounts of less than 160, less than 170, less than 180, less than 190 and less than 210, less than 220, less than 230 and less than 240 mg / m2 are included. For example, the person skilled in the art will expect that concomitant administration with corticosteroids or antibiotics will allow slightly higher doses of the immunoconjugate even in cases of side effects on the skin, which however can be easily evaluated by the person skilled in the art. .

To evaluate the success of the administration of a drug, here an immunoconjugate (its ability to produce a functional response, ie its efficacy), different "responses" to an administration are distinguished.

In the context of M and other plasmaproliferative diseases, the responses are distinguished as follows: the term "complete response" refers to the negative immunofixation of serum and urine and disappearance of any soft tissue plasmacytomas and < 5% of plasma cells in bone marrow; the term "severe complete response" (sCR = Stringent Complete Response) refers to CR as defined above plus the normal FLC ratio and absence of clonal cells in bone marrow by immunohistochemistry or immunofluorescence; The expression very good partial response (VGPR = Very Good Partial Response) refers to component M in serum and urine detectable by immunofixation, but not in electrophoresis or > 90% or greater reduction in M component of serum plus M component of urine < 100 mg per 24h; The term partial response (PR = Partial Response) refers to > 50% reduction of M protein in serum and reduction in 24 hours of urinary M protein by > 90% or a < 200 mg per 24 hours, if the serum M protein and urine are not measured, a decrease of > 50% in the difference between FLC levels involved and not involved is required instead of the M protein criteria, if the M protein in serum and urine can not be measured, and the assay or analysis of free light chains in serum, reduction of > 50% in bone marrow plasma cells is required in place of protein, provided the percentage of baseline was > 30%, in addition to the above criteria, if present in the reference line, reduction of > 50% in size of soft tissue plasmacytomas is also required (Durie et al., 2006).

The term "minor response" (MR = Minor Response) in relation to patients with relapse / refractory myeloma refers to > 25% but reduction of < 49% of protein M in serum and reduction in protein M of urine of 24 hours in 50-89%, which still exceeds 200 mg for 24 hours, in addition to the above criteria, if present in the reference line, reduction of 25 -49% in size of soft tissue plasmacytomas is also required, without increase in size or number of lithic bone lesions (developed by compression fracture does not exclude response).

However, a response, although not formally classified, also includes a reduction of at least 30%, preferably at least 40% or 50% in serum FLC levels. This is particularly significant in cases where the M protein can not be measured.

The expression stable disease (SD = Stable Disease) refers in the context of plasmaproliferative diseases of the present invention, by not meeting the criteria for CR, VGPR, PR or progressive disease while the expression progressive disease (PD = Progressive Disease) refers to the 25% increase from the lowest response value in any one or more of the following: - Component M in serum (absolute increase should be> 0.5g / 100 ml) and / or - Component in urine (absolute increase should be> 200 mg for 24 hours) and / or - Only in patients without M protein levels measurable in serum and urine; the difference between FLC levels involved and not involved (absolute increase must be> 100 mg / 1) - Percentage of plasma cells in bone marrow (absolute percent should be> 10%) - Defined development of new bone lesions or soft tissue plasmacytomas or defined increase in the size of existing bone lesions or soft tissue plasmacytomas.

-Development of hypercalcemia (corrected serum calcium> 11.5mg / 100ml) that can only be attributed to the proliferative disorder of plasma cells.

The term "myeloma with relapse" refers here to an active form of M in a subject, wherein the subject underwent at least one prior treatment regimen and did not meet the criteria for relapse / refractory myeloma.

The term refractory myeloma in general refers to a condition of the disease when the number of plasma cells continues to increase even when treatment is provided, ie the disease at the time of evaluation has shown that it is not capable of receiving the treatment regimen administered .

The term relapsed / refractory myeloma refers here to the relapse of disease while in rescue therapy, or progression within 60 days of the most recent therapy.

The term refractory phenotype includes any type of refractory myeloma, this is refractory and relapse / refractory myeloma.

The expression relapse or refractory myeloma covers refractory relapse and relapse / refractory myeloma.

In the clinical study discussed in more detail below, subjects have been treated with at least one immunomodulator and one proteasome inhibitor therapy, which have failed before entering the study. The disease was considered refractory to treatment if the subject experienced progressive disease (PD) in his previous regimen.

The expression "progression to", for example "active MM" in relation to patients with SMM refers in the context of the present invention to evidence of progression based on the criteria of the international myeloma working group (IMWG = International Myeloma Working Group) for progressive disease in MM in any one or more of the following that feels related to the proliferative disorder of underlying clonal plasma cells, development of new soft tissue plasmacytomas or bone lesions, hypercalcemia (> 11 mg / 100 ml), decrease in hemoglobin of > 2 g / 100 ml, and serum creatinine level = 2 mg / 100 ml. (Kyle &Rajkumar, 2009).

The pathogenesis of multiple myeloma involves binding of myeloma cells, via cell surface adhesion molecules, to bone marrow stromal cells (BMSCs) as well as the extracellular matrix (ECM = Extracellular Matrix). This active link, and in this way can ultimately be made responsible for growth of multiple myeloma cells, drug resistance, immigration of MM cells in the bone marrow medium (Munshi et al., 2008). In particular, the adhesion of multiple myeloma cells to ECM by syndecan-1 (CD138) to type I collagen, induces the expression of matrix metalloproteinase 1, thereby promoting bone resorption and tumor invasion (Hideshima et al. 2007). Interactions between multiple myeloma cells and the bone marrow microenvironment results in activation of a proliferative and antiapoptotic pleyotropic cascade.

For multiple myeloma patients, but also for patients suffering from other diseases associated with bone pain, there are a number of supportive treatments to treat this and other symptoms. Appropriate medications include bisphosphonates (eg, pamidronate, zoledronic acid), which can slow down bone damage. It has also been shown that these agents are capable of reducing osteolytic bone lesions and avoiding fractures (Ludwig et al., 2007). Primarily they are delivered through a vein to decrease the risk of bone complications such as fractures, and to reduce abnormally high blood calcium levels (hypercalcemia). Data suggest that bisphosphonates reduce bone pain associated with MM. Patients can also undergo surgery if their bones are weak or broken.

In one embodiment, immunoconjugates reduce, in particular reduce to an acceptable level, bone pain and / or bone complications, such as osteonecrosis. A reduction to an acceptable level involves in particular the ability to interrupt the administration of a medication that alleviates these pains or is aimed at reducing these bone complications. Bisphosphonates such as pamidronate, zoledronic acid and clodronate are commonly administered to alleviate bone complications, such as osteonecrosis in MM patients and thus to relieve bone pain associated with such complications. Common bisphosphonates include for oral administration FOSOMAX, BONIVA, ACTONEL, DIDRONEL and SKELID, for intravenous administration, BONEFOS, AREDIA and ZOMETA.

A reduction in bone pain and / or bone complications according to the present invention may result in a reduction in the amount of pain medication that is administered to a patient and / or in the amount of any drugs that are administered to counterattack these complications This reduction can be (i) with respect to a previously administered amount, when the patient undergoes (a) a treatment of the disease that causes bone pain and / or bone complications that differ from treatment according to the present invention or (b) without treatment or (ii) with respect to an amount administered to another patient suffering from the same disease and is in approximately the same stage of the disease. Said preference reduction is approximately 10%about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% reduction and preferably a complete cessation of the administration of the drug. When the latter is administered this is, when the patient does not subjectively require medication against bone pain and / or bone complications as a result of being administered the immunoconjugate according to the present invention, either alone or as part of a anticancer combination according to the invention, the administration of the immunoconjugate is said to have reduced bone pain and / or bone complications to an acceptable level. As a result, adverse side effects resulting from the medication administered to relieve bone pain and / or bone complications should be reduced or abolished.

Following the migration of multiple myeloma cells to the bone marrow stromal compartment, adhesion between multiple myeloma cells and B SCs upregulates many cytokines such as interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF- 1) they have. growth activities of .tumor and angiogenic (Hideshima et al., 2007). The signaling cascades initiated by this cytokine eventually result in resistance of M cells to conventional therapeutics (Anderson et al., 2000, Hideshima et al., 2006).

In the normal human hematopoietic compartment, the expression of CD138 is restricted to plasma cells (Wijdenes, 1996, Chilosi, 1999) and CD138 is not expressed in peripheral blood lymphocytes, monocytes, granulocytes and red blood cells. In particular, CD34 + stem and progenitor cells do not express CD138 and anti-CD138 mAbs do not affect the number of colony forming units in hematopoietic stem cell cultures (ijdenes, 1996). In non-hematopoietic compartments, CD138 is expressed primarily in simple and stratified epithelia within the lung, liver, skin, kidneys and intestines. Only weak staining was seen in endothelial cells (Bernfield, 1992; Vooijs, 1996). It has been reported that CD138 exists in polymorphic forms in human lymphoma cells (Gattei, 1999).

Monoclonal antibodies B-B4, BC / B-B4, B-B2, DL-101, 1 D4, MI15, 1.BB.210, 2Q1484, 5F7, 104-9, 281-2 in particular B-B4 have been reported that are specific to CD138. Of those B-B4, 1D4 and MI15 recognize both the intact molecule and the core protein of CD138 and were shown to recognize any of the same or closely related epitopes (Gattei, 1999). Previous studies reported that B-B4 does not recognize soluble CD138, but only CD138 in the membrane-bound form (Wijdenes, 2002).

The initial anti-CD138 antibody was developed by Diaclone SAS (Besan on, France) as the murine parental ab B-B4, generated by immunization with the human multiple myeloma cell line U266, using standard hybridoma technology (Clement, 1995; Wijdenes , nineteen ninety six) . B-B4 binds to a linear epitope between residues 90-93 of the core protein in human syndecan-1 (CD138) (Wijdenes, 1996; Dore, 1998). Consistent with the expression pattern of CD138, B-B4 is shown to react strongly with the RPMI8226 plasma cell line, but does not react with endothelial cells. Also consistent with the expression pattern of CD138, B-B4 also reacts with A431 epithelial cell lines (derived from keratinocyte) and HepG2 (hepatocyte derivative). An immunotoxin B-B4-saporin was also highly reactive towards the plasma cell line RPMI8226, in fact considerably more toxic than free saporin. However, of the two epithelial cell lines tested, B-B4-saporin shows only toxicity towards the A431 cell line, although in a clonogenic assay B-B4-saporin showed no inhibitory effect on A431 cell outgrowth (Vooijs, 1996) . Other investigators reported the lack of specificity of antigens associated with MM against tumors (Couturier, 1999).

B-B4 covalently linked to the maytansinoid DM1 showed selective cytotoxicity in cell lines and multiple myeloma cells, as well as anti-cancer activity in human multiple myeloma xenograft models in SCID mice (Tassone, 2004).

The present invention uses the term "tumor cell" to include cancer cells as well as precancerous cells which may or may not be part of a solid tumor.

An agent directed according to the present invention is capable of associating with a molecule expressed by a target cell and includes peptides. In particular, agents directed in accordance with the present invention include targeted antibodies and molecules not targeted to immunoglobulin, which can be based on proteins without immunoglobulin including but not limited to molecules AFFILIN®, ANTICALINS® and AFFIBODIES®. Molecules not directed to immunoglobulin also include non-directed peptide molecules such as DNA and RNA directed oligonucleotides (aptamers), but also physiological ligands, in particular ligands of the antigen in question such as CD138.

An antibody, directed according to the present invention is either based on a natural antibody or is produced synthetically or by genetic engineering and binds an antigen in a cell or cells (cell or targeted cells) of interest. An antibody directed in accordance with the present invention includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody) or an antibody fragment. The targeted antibody can be engineered, for example, to improve its affinity to target cells (Ross, 2003) or decrease its immunogenicity. The targeted antibody can be ligated to a liposomal formulation including effector molecules (Carter, 2001). An antibody fragment comprising a portion of an intact antibody, preferably the variable or antigen binding region of the intact antibody. Examples of antibody fragments according to the present invention include Fab, Fab ', F (ab') 2 / and Fv fragments but also diabodies; domain antibodies (dAb) (ard, 1989, U.S. Patent Number 6,005,079); linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. In a single-chain variable fragment (scFv) antibody the heavy and light chains (VH and VL) can be linked by a short amino acid linker having for example the sequence (glycine4serine) n, which has sufficient flexibility to allow both domains assemble a functional antigen binding cavity. The addition of various signal sequences may allow a more precise targeting of the targeted antibody. The addition of the light chain constant region (CL) can allow dimerization by disulfide bonds, giving increased stability and avidity. Variable regions for constructing scFv can, if a mAb against a target of interest is available, be obtained by RT-PCR that clones the variable regions of mRNA that is extracted from the precursor hybridoma. Alternatively, scFv can be generated de novo by phage display technology (Smith, 2001). As used herein, the term "functional fragment" when used with reference to a directed antibody, is intended to refer to a portion of the engineered antibody that is capable of specifically binding to an antigen that is specifically bound by the antibody to which it makes reference. A bispecific antibody according to the present invention may for example have at least one arm that is reactive against a target tissue and an arm that is reactive against a linker portion (U.S. Patent Publication Number 20020006379). A bispecific antibody according to the present invention can also bind to more than one antigen in a target cell (Carter, 2001). An antibody according to the present invention can be modified for example by introducing cysteine residues to introduce thiol groups (Olafsen, 2004).

According to the present invention, the targeted antibody can be derived from any source and can be but is not limited to a camel antibody, a murine antibody, a chimeric mouse / human antibody or a chimeric monkey / human antibody, in particular a chimeric mouse / human antibody such as nBT062.

Humanized antibodies are antibodies that contain sequences derived from a human antibody and a non-human antibody and are also within the scope of the present invention. Suitable methods for humanizing antibodies include CDR grafting (complementarity determination region graft) (EP 0 239 400; WO 91/09967; US Patents Nos. 5,530,101; and 5,585,089), restoration with veneers or coating prostheses (EP 0 592 106; EP 0 519 596; Padlan, 1991; Studnicka et al., 1994; Roguska et al., 1994), chain exchange (U.S. Patent Number 5,565,332) and Delmmunosation ™ (Biovation, LTD). In CDR grafting, the mouse complementarity determining regions (CDRs) for example of mAb B-B4 are grafted onto human variable structures, which are then bound, to human constant regions, to create a human B-B4 antibody (hB- B4). Several antibodies humanized by CDR grafting are now in clinical use, including MYLOTARG (Sievers et al., 2001) and HECEPTIN (Pegram et al, 1998).

The coating prosthesis technology utilizes a combination of molecular modeling, statistical analysis and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble known antibody surfaces of the target host. Strategies and methods for antibody coating, and other methods for reducing immunogenicity of antibodies within a different host, are described, for example, in U.S. Pat. Number 5,639,641. Human antibodies can be made by a variety of methods known in the art including phage display methods. See also U.S. Patents Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and publications of international patent applications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

Targeted antibodies that have undergone any non-natural modification such as chimeric mouse / human antibodies or chimeric monkey / human antibodies, humanized antibodies or antibodies that were designed by "example to improve their affinity to target cells or decrease their immunogenicity but also to antibody fragments in particular functional fragments of these targeted antibodies which have undergone any unnatural modification, diabodies, domain antibodies, linear antibodies, single chain antibody molecules where, and multispecific antibodies are referred to herein as targeting antibodies of engineering.

Chimerized antibodies maintain the antibody binding region (ABR or Fab region) of the non-human antibody, for example the murine antibody on which they are based, while any constant regions can be provided for example by a human antibody. In general, the chimerization and / or exchange of constant regions of an antibody will not affect the affinity of an antibody because the regions of the antibody that contribute to antigen binding are not affected by this exchange. In a preferred embodiment of the present invention, the engineered antibody, in particular chimerized of the present invention, may have a higher binding affinity (as expressed by KD values) than the respective non-human antibody on which it is based. In particular, the nBT062 antibody and antibodies based thereon may have higher antibody affinity than murine B-B4.

In another preferred embodiment of the present invention, immunoconjugates comprising those engineered / chimerized antibodies also exhibit this superior antibody affinity. These immunoconjugates may also exhibit in other embodiments, other advantageous properties, such as a higher tumor burden reduction than their counterparts containing B-B4. In a preferred embodiment, engineered engineered antibodies in particular chimerized ones exhibit binding affinities that are characterized by KD dissociation constants (nM) less than 1.6, less than 1.5 or about or less than 1.4, while their murine counterparts are characterized by Dissociation constants KD (nM) of approximately or greater than 1.6. Immunoconjugates comprising targeting agents such as targeted antibodies can be characterized by KD dissociation constants (nM) of less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2.0, less or about 1.9 are preferred, whereas immunoconjugates comprising the murine counterpart antibodies can be characterized by KD dissociation constants (nM) of about or greater than 2.6 (compare Table 9, Materials and Methods).

The basic antibody molecule is a bifunctional structure in which the variable regions bind antigen while the remaining constant regions can produce antigen-independent responses. The major classes of IgA antibodies, IgD, IgE, IgG and IgM, are determined by the constant regions. These classes can also be divided into subclasses (isotypes). For example, the IgG class has four isotypes, ie, IgGl, IgG2, IgG3, and IgG4 that are determined by the constant regions. Of the various classes of human antibodies, only human IgGl, IgG2, IgG3 and IgM are known to effectively activate the complement system. While the constant regions do not form the antigen binding sites, the structure or arrangement of the constant regions and hinge region can confer segment flexibility in the molecule allowing it to bind to the antigen.

Different IgG isotypes can bind to receptors Fe in cells such as monocytes, B cells and NK cells, thereby activating the cells to release cytokines. Different isotypes can also activate complement, resulting in local or systemic inflammation. In particular; the different IgG isotypes can bind FcyR to different degrees. FcyRs are a group of surface glycoproteins that belong to the Ig super family and are expressed primarily in leukocytes. The glycoproteins. FcyR are divided into three classes designated FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16). While IgGl, IgG2 and IgG3 bind strongly to a variety of these classes of glycoproteins FcyR, IgG4 exhibits a much weaker bond. In particular, IgG4 is an intermediate linker FcyRI, which results in relatively low or even null antibody dependent cell cytotoxicity (ADCC = Antibody Dependent Cellular Cytotoxicity), and does not bind to FcyRIIIA or FcyRIIA. IgG4 is also a weak linker of FcyRIIB, which is an inhibitory receptor. In addition, IgG4 mediates only weakly or null complement fixation and weak or no complement-dependent cytotoxicity F (CDC). In the context of the present invention, IgG4 can be specifically employed to prevent Fe-mediated directing of hepatic FcR since it exhibits no interaction with FCRYII in liver sinusoidal endothelial cells (LSECs = Liver Sinusoidal Endothelial Cells), no or weak interaction with FcRyl -III in Kupffer cells (macrophages) and no interaction with FcRylH in liver NK cells. Certain mutations that further reduce any CDC are also part of the present invention. For example, IgG4 residues at positions 327, 330 and 331 were shown to reduce antibody-dependent cellular cytotoxicity (ADCC = Antibody Dependent Cellular Cytotoxicity) and CDC (Amour, 1999; Shields, 2001). - One of more mutations that stabilize the antibody are also part of the present invention (also referred to herein as "stabilizing mutations"). These mutations include in particular mutations of leucine-to-glutamic acid in the CH2 region of IgG4 and serine-to-proline exchange in the hinge nucleus IgG4. These mutations decrease, in certain embodiments of the invention, the amount of half molecules to less than 10%, less than 5% and preferably less than 2% or 1%. Furthermore, the in vivo half life of stabilized antibodies can be increased several days including 1, 2, 3, 4 or more than 5 days (Schuurman, 1999).

When the present invention relates to an immunoconjugate comprising an engineered engineered antibody that confers IgG4 isotype properties, this means that engineered antibody exhibits significantly reduced affinity to cells expressing Fe receptor as compared to the affinity of isotype antibodies. IgGl. These properties are preferably conferred by an additional antibody region, which is different from ABR, where the additional antibody region in whole or in part is from a human antibody. The result is an equally small deficiency (greater than 90% with respect to its IgG1 isotype counterpart) or a total potential for inducing CDC or ADCC compared to the potential to induce CDC or ADCC usually observed with IgGl isotype antibodies. This property can be measured in cell-based assays by employing the engineered antibody engineered in its unconjugated form. CDC and ADCC can be measured by different methods such as that described in Cancer Immunol. Immunother., 36, 373 (1993) or the GUAVA Cellular Toxicity Assay. The overall benefit of immunoconjugates comprising at least part of an engineered engineered antibody that confers IgG4 isotype properties is an improvement in binding specificity and reduced toxicity. Also, the resulting reduced affinity to Fe receptors improves the antigen-specific target of tumor cells which leads to reduced toxicity against CD138 negative cells.

Targeted agents, including directed antibodies described herein may also be described or specified in terms of their antigen binding affinity, in particular to CD138. Preferred binding affinities of targeted agents such as targeted antibodies are characterized by KD dissociation constants (nM) less than 1.6, less than 1.5 or about or less than 1.4. For immunoconjugates comprising targeted agents such as antibodies directed with KD dissociation constants (nM) less than 1.6, less than 1.5 or less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2.0 , less than or about 1.9, are preferred.

An antigen binding region (ABR) according to the present invention will vary based on the type of engineered antibody or engineered engineered antibody employed. In an antibody of natural origin and in the majority of chimeric and humanized antibodies, the antigen binding region is constituted by a light chain and the first two domains of a heavy chain. However, in heavy chain antibody lacking light chains, the antigen binding region will be constituted for example of the first two domains of the heavy chain only, while in single chain antibodies (ScFv), which combine in a single Polypeptide chain The heavy and light chain variable domains of an antibody molecule, ABR is provided by only one polypeptide molecule. FAB fragments are usually obtained by digestion with papain and have a light chain and part of a heavy chain and thus comprise an ABR with only one antigen combining site. On the other hand, diabodies are small antibody fragments with two antigen binding regions. In the context of the present invention, however, an antigen binding region of a directed antibody or engineered antibody is any region that primarily determines the binding specificity of the engineered antibody or engineered antibody.

If the ABR or other targeted antibody region is said to be "of a certain antibody", for example a human or non-human antibody, this means in the context of the present invention that ABR is already identical to an ABR of corresponding natural origin. or it is based on it. An ABR is based on an ABR of natural origin if it has the binding specificity of the naturally occurring ABR. However, said ABR may comprise, for example, point mutations, additions, deletions or post-translational modification such as glycosylation. This particular ABR can have more than 70%, more than 80%, more than 90%, preferably more than 95%, more than 98% or more than 99% sequence identity with the ABR sequence of natural origin . nBT062 (see also Figure 1) is a murine human chimeric IgG4 mAb, ie a chimerized version of B-B4. This chimerized version of B-B4 was created to reduce the humoral response of human anti-mouse antibodies (HAMA = Human Anti- ouse Antibody) while maintaining the functionality of the antibody binding region of B-B4 for CD138. Surprisingly, the results obtained using an immunoconjugate comprising this engineered engineered antibody were much more homogeneous (the variance in the results was reduced). The protocol to produce nBT062 is specified below. Chinese hamster ovary cells expressing nBT062 have been deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1, D-38124 Braunschweig on December 11, 2007. The identification number is DS ACC2875. A specific chimeric antibody CD138 based on B-B4 is generically referred to herein as C-B-B4.

The amino acid sequence for both the heavy and light chains has been predicted from the translation of the nucleotide sequence for nBT062. The predicted amino acid sequence for heavy chain and light chain are presented in Table 3. Predicted variable regions are with bold, predicted CDRs are underlined.

Table 3. Predicted Amino Acid Sequence for nBT062 - Predicted sequence of heavy chain nBT062 (SEQ ID NO: l): 1 QVQLQQSGSE LMMPGASVKI SCKATGYTPS NYWIEWVKQR PGHGLEWIGE 51 ILPGTGRTIY NEKFKGKATF TADISSNTVQ MQLSSLTSED SAVYYCARRD 101 YYGNFYYAMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL 151 VKDYFPEPVT VS NSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT 201 KTYTCNVDHK PSNTKVDKRV ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK 251 DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS 301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV 351 YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 401 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQKSLSLSLG (K) C-terminal lysine tends to cut and may be present due to incomplete clipping in a certain proportion. (K) in parentheses is not part of SEQ ID NO: l. - Predicted sequence of light chain nBT062 (SEQ ID NO: 2): 1 DIQMTQSTSS LSASLGDRVT ISCSASQGIN NYLNWYQQKP DGTVELLIYY 51 TSTLQSGVPS RFSGSGSGTD YSLTISNLEP EDIGTYYCQQ YSKLPRTFGG 101 GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG RGEC 201 LSSPVTKSFN Table 4. Shows a comparison of the general CDR definitions of Kabat and Chothia and the predicted CDRs for nBT062.

CDR definition of Kabat ??? 062 CDR1 light chain: CDR1 residues: waste 24-34 24-34 CDR2: CDR2 residues: residues 50-56 50-56 CDR3: CDR3 waste: waste 89-97 89-97 CDR1 heavy chain: CDR1 waste: waste 31-35 31-35 CDR2: CDR2 residues: residues 50-56 51-68 CDR3: CDR3 waste: waste 95-1.02 99-111 CDR definition of Chothia nBT062 CDR1 light chain: CDR1 residues: waste 26-32 24-34 CDR2: CDR2 residues: residues 50-52 50-56 CDR3: CDR3 waste: residues 91-96 89-97 CDR1 heavy chain: CDR1 waste: waste 26-32 31-35 CDR2: CDR2 residues: residues 52-56 51-68 CDR3: CDR3 residues: residues 96-101 99-111 Fully human antibodies can also be used. These antibodies can be selected by the phage display approach, wherein CD138 or its antihygienic determinant is used to selectively bind phage display, for example B-B4 variable regions (see, Krebs, 2001). This approach is advantageously coupled with an affinity maturation technique to improve the affinity of the antibody. All the antibodies referred to herein are isolated antibodies (see U.S. Patent Publication Number 20090175863).

In one embodiment, the directed antibody in its unconjugated form is internalized moderately or poorly. Moderate internalization constitutes approximately 30% to approximately 75% total antibody internalization, deficient internalization constitutes approximately 0.01% up to approximately 30% internalization after 3 hours of incubation at 37 ° C. In another preferred embodiment, the directed antibody binds to CD138, for example antibodies B-B4, BC / B-B4, B-B2, DL-101, 1 D4, MI15, 1.BB.210, 2Q14S4, 5F7, 104- 9, 281-2 in particular B-B4. Hybridoma cells that were generated by hybridizing SP02 / 0 myeloma cells with spleen cells from Balb / c mice were deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1, D-38124 Braunschweig on December 11, 2007 . The identification number of these hybridoma cells expressing B-B4 is DS ACC2874. In another embodiment, the engineered antibody does not substantially bind CD138 not expressed on the cell surface. When, in the context of the present invention, the name of a specific antibody is combined with the expression "directed antibody" such as "directed antibody nBT062", this means that this targeted antibody has the binding specificity of the nBT062 antibody. If a directed antibody is said to be "based on" a specified antibody, this means that this targeted antibody has the binding specificity of this antibody, but can take any form consistent with the previous description of a targeted antibody. When, in the context of the present invention, the name of a specific antigen is combined with the expression "directed antibody" such as "CD138 targeted antibody", this means that this targeted antibody has binding specificity for CD138. If, in the context of the present invention, for example a "targeted" antibody is said to do something "selectively" such as "selectively targeted to a cell surface that expresses CD138" or that is "selective" for something, this means that there is a significant selectivity (ie a higher affinity towards CD138 positive cells compared to CD138 negative cells), so that in the case of the example provided, CD138 expressed on cell surface compared to any other antigen expressed on cell surface. Adverse side effects in a given environment can be substantially reduced or even avoided due to this selectivity. "directed epigens without immunoglobulin" according to the present invention, include directed molecules derived from non-immunoglobulin proteins as well as non-peptide directed molecules. Small proteins without immunoglobulin that are included in this definition are designed to have specific affinities towards, in particular CD138 expressed on the surface. These small proteins without immunoglobulin include scaffold-based engineering molecules such as Affilin® molecules that have a relatively low molecular weight such as between 10 kDa and 20 kDa. Scaffolds or appropriate structures include for example crystalline range. Those molecules have in their natural state non-specific binding activity towards the target molecules. By engineering the protein surface through locally defined randomization of amino acids exposed to solvent, completely new binding sites are created. Previous unbound proteins in this manner are transformed into specific binding proteins. These molecules can be specifically designed to bind a target, such as CD138, and allow specific delivery of one or more effector molecules (see scil Proteins GmbH at www.scilproteins.com, 2004). Another type of molecules directed without immunoglobulin are derived from lipocalins, and include for example ANTICALINS®, which resemble in structure something to immunoglobulins. However, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues. The binding cavity of lipocalins can be reconfigured to recognize a molecule of interest with high affinity and specificity (see, for example, Beste et al., 1999). Artificial bacterial receptors such as those marketed under the brand name Affibody® (Affibody AB) are also within the scope of the present invention. These artificial bacterial receptor molecules are simple small proteins and can be composed of a three-helix beam based on the scaffold of one of the IgG binding domains of Protein A (Staphylococcus aureus). These molecules have similar binding properties to many immunoglobulins but are substantially smaller, have a molecular weight that often does not exceed 10kDa and are also comparably stable. Suitable artificial bacterial receptor molecules are for example described in U.S. Pat. Nos. 5,831,012; 6,534,628 and 6,740,734.

Other "directed molecules without immunoglobulin" are physiological ligands of the antigen in question. Physiological ligands of CD138 include but are not limited to ADAMTS4 (aggrecanase-1), antithrombin-3, bFGF, cathepsin G, CCL5 (RANTES), CCL7, CCL11, CCL17, CD44, collagens (collagen type 1, collagen type 2) , type 3 collagen, type 4 collagen, type 5 collagen, type 6 collagen), CXCL1, elastase, gpl20, hepatocyte growth factor [HGF = Hepatocyte Growth Factor], laminin-1, laminin-2, laminin-5, midkine , MMP-7, neutrophil elastase and pleiotropin (HBNF, HBGF-8). Non-peptide directed molecules include but are not limited to DNA and RNA oligonucleotides that bind to CD138 (aptamers).

An "effector molecule" according to the present invention is a molecule or a derivative or its analog that binds to a targeted agent, in particular a targeted antibody and / or an engineered engineered antibody, and which exerts a desired effect, example apoptosis or another type of cell death, or a brake of the continuous cell cycle in the targeted cell (s). Effector molecules according to the present invention include molecules that can exert the desired effects in a target cell and include but are not limited to cytotoxic drugs, including low molecular weight cytotoxic drugs (molecular mass less than 1500 Da, preferably less than 1400 , less than 1200, less than 1000, less than 800, less than 700, less than 600, less than 500, less than 300 but in general greater than 120 Da). These cytotoxic drugs according to the present invention are in general non-proteinaceous biological cytotoxic drugs and contain or induce, before administration, the production of another cytotoxic drug of at least 5 C atoms, 10 C atoms, preferably more than 12 C atoms, often greater than 20 C atoms and sometimes greater than 30, 40 or 50 CY atoms in general at least one ring structure, such as a benzene ring which is often substituted. However, interconnect ring structures are often part of these molecules. These non-proteinaceous biological cytotoxic drugs can be interspersed into DNA (DNA intercalators) or DNA alkylation, inhibiting microtubule formation are inhibitors of mitosis. The inhibitors of enzymes involved in the structural integrity of DNA, such as acetylate histone or inhibitors of enzymes that are otherwise vital to a cell and cause breakdown of cellular metabolism. Effectors can also be categorized as radionuclides, biological response modifiers, pore-forming agents, ribonucleases, apoptotic signaling cascade proteins with apoptosis-inducing activities, antisense oligonucleotides, anti-metastasis agents, antioxidants, antibodies or cytokines, as well as their functional derivatives or analogues / fragments.

Toxins may include bacterial toxins, such as but not limited to Diphtheria or Exotoxin A toxin, plant toxins, such as but not limited to Ricin, other alkaloids and polyphenols, mycotoxins such as alpha amanitin or more especially Amatoxins and Phaltoxins. Toxins can not only be of bacterial origin but also fungal, of plants, vertebrates and of invertebrate origin, all of which can be modified in genetic or chemical form. Furthermore, the toxins can also be environmental toxins such as but not limited to methyl mercury. Effector molecules can be proteins such as those of apoptotic signaling cascades with activities that induce apoptosis, including but not limited to Granzima B, Granzima A, Caspase-3, Caspase-7, Caspase-8, Caspase-9, Bid truncate- ( tBid), Bax and Bak. The toxins can also be dolastatins 10 and 15 are small peptides isolated from sea wedge hare (Dolabella auricularia) that has been shown to interact with tubulin.

Effector Molecular mass (g / mol [Da] Doxorubicin 564 Danurubicin 528 Vinblastine 811 Docetaxel 808 Paclitaxel 854 Epothilone B 508 Vorinostat 264 Neocarzinostatin 660 Calicheamicin ?? 1368 Esperamycin 1342 Methotrexate 454 Components of 482 Silymarin asoprocol 302 Acid 132 aminolevulin Miltefosina 407 Epigallocatechin 459 gallate (EGCG) Psoraleno 186 elfalan 304 Table 5 provides examples of low molecular weight cytotoxic drugs that can serve as effector molecules.

In a preferred embodiment, the effector molecule increases the internal effector supply of the immunoconjugate, particularly when the natural form of the antibody on which the targeted antibody of the immunoconjugate is based is poorly internalizable. In another preferred embodiment, the effector in its native form is not selective. In certain embodiments, the effector has high non-selective toxicity, including systemic toxicity, when it is in its native form. The "native form" of an effector molecule of the present invention is an effector molecule before binding to the targeted agent to form an immunoconjugate. In another preferred embodiment, the non-selective toxicity of the effector molecule is substantially eliminated upon conjugation with the targeted agent. In another preferred embodiment, the effector molecule causes, upon reaching the target or target cell, death or brake of the cell cycle, including continuous cell cycle brake, in the target cell.

An effector molecule according to the present invention includes but is not. limited to antineoplastic agents, in particular intracellular chemotherapeutic agents defined below.

Low molecular weight cytotoxic drugs (see above for molecular weight) may be preferably antimitotic, more particular agents that affect tubulin, including tubulin polymerization inhibitors such as maytansinoids, } dolastatins (and derivatives such as auristatin) and cryptophycin and potent taxoid drugs (taxane) (Payne, 2003). It is also included in the definition of small drug of high toxicity and there are other agents that interfere with tubulin such as epothilones (for example ixabepilone) and colchicine derivatives (agents that interfere with tubulin are discussed further below).

An effector molecule which is a maytansinoid, includes maytansinoids of any origin, including but not limited to synthetic maytansinol and analogs and maytansinol derivatives.

Maytansine is a natural product originally derived from the Ethiopian bush Maytenus serrata (Remillard, 1975, U.S. Patent Number 3,896,111). This drug inhibits the polymerization of tubulin, resulting in mitotic block and cell death (Remillard, 1975, Bhattacharyya, 1977, Kupchan, 1978). The cytotoxicity of ma-itansin is 200-1000 times higher than that of anticancer drugs in clinical use that affect the polymerization of tubulin, such as Vinca alkaloids or taxol. However, clinical trials of maytansine indicate that it lacks a therapeutic window due to its high systemic toxicity. Maytansine and maytansinoids are highly cytotoxic but their clinical use in cancer therapy has been greatly limited by the severe systemic side effects primarily attributed to their poor selectivity for tumors. Clinical trials with maytansine show serious adverse effects on the central nervous system and the gastrointestinal system.

Maytansinoids have also been isolated from other plants including seed tissue of Trewia nudiflora (U.S. Patent Number 4,418,064).

Certain microbes also produce maytansinoids, such as maytansinol and C-3 maitansinol esters (U.S. Patent Number 4,151,042).

The present invention is directed to maytansinoids of any origin, including synthetic maytansinol and maytansinol analogues which are described, for example, in U.S. Pat. Numbers 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,371,533; 4,424,219 and 4,151,042.

In a preferred embodiment, the maytansinoid is a thiol containing maytansinoid and is more preferably produced according to the processes described in US Pat. Number 6,333,410 granted to Chari et al. or in Chari et al. (Chari, 1992).

DM-1 (N2-deacetyl-N2- (3-mercapto-l-oxopropyl) -maitansine) is a preferred effector molecule in the context of the present invention. DM1 is 3 to 10 times more cytotoxic than maytansine and has been converted to a prodrug by ligating it by one or several disulfide bonds to a monoclonal antibody directed towards an antigen associated with tumor. Certain of these conjugates (sometimes called "tumor activated drugs" (TAPs = Tumor Activated Prodrugs)) are not cytotoxic in the blood compartment, since they are activated by associating with the target cells and internalizing, thereby releasing the drug ( Bláttler, 2001). Several antibody-DMl conjugates have been developed (Payne, 2003), and have been evaluated in clinical trials. For example, treatment with huC242-DMl in patients with colorectal cancer was well tolerated, did not induce any immunodetectable response and had a prolonged circulation time (Tolcher, 2003).

Other particularly preferred maytansinoids comprise a side chain containing a hindered sterically hindered thiol such as but not limited to maytansinoids N2 '-deacetyl-N2'- (4-mercapto-l-oxopentyl) -maitansine, also referred to as "DM3" and N2 '-deacetyl-N2'- (4-methyl-4-mercapto-l-oxopentyl) -maitansine, also referred to as "DM4". The synthesis of DM4 is illustrated in Figures 3 and 4 and is described elsewhere here. DM4 differs from DM1 and DM3 since it has methyl groups in its cxC. This results in a spherical hindrance when DM4 is linked by a particular linker, but not limited to, a linker comprising a disulfide bond, to a targeted agent such as nBT062. A wide variety of maytansinoids containing a sterically hindered thiol group (which possesses one or two substituents, in particular alkyl substituents, such as the methyl substituents of DM4) are described in U.S. Patent Publication. Number 2004/0235840, published on November 25, 2004, which is incorporated herein in its entirety by reference. The steric hindrance conferred by alkyl groups such as the methyl groups on the carbon adjacent to the sulfur atom of DM3 and DM4, can affect the intracellular cleavage rate of the immunoconjugate. The variable alkyl unit therefore affects potency, efficacy and safety / toxicity in vitro and in vivo.

As reported by Goldmahker et al., In the U.S. Patent Publication. Number 2006/0233814, such impairment, induces alkylation (eg, methylation) of the free drug, once the drug is released into its target. Alkylation can increase the stability of the drug allowing the so-called non-specific activation effect (bystander). However, as will be appreciated by the person skilled in the art, other effector molecules comprising substituents such as alkyl groups at positions that result in a steric hindrance when the effector is linked to a target agent by a linker, are part of the present invention. (U.S. Patent Publication Number 2004/0235840). Preferably, this hindrance induces a chemical modification such as alkylation of the free drug to increase its total stability, which allows the drug not only to induce cell death or continuous cell cycle brake in tumor cells expressing CD138 but optionally also affects cells auxiliaries that for example support 0 protect the tumor from drugs, particularly tumor stromal cells and tumor vasculature, and that generally do not express CD138 to diminish or lose their support or protection function.

Maytansine was evaluated in clinical trials Phase 1 and Phase II under the auspices of the National Cancer Institute (NCI = National Cancer Institute) under IND # 11,857 (submitted to the FDA on September 19, 1975). Both complete and partial responses were seen in patients with hematologic malignancies and partial responses in patients with a broad spectrum of solid tumors (Blum and Kahlert., 1978, Issell and Crooke, 1978, Chabner et al., 1978, Eagan et al., 1978, Cabanillas et al., 1978). However, significant toxicities including nausea, vomiting, diarrhea, elevations of liver function tests, lethargy and peripheral neuropathy were noted (see Maytansine IND # 11,857, Annual Report, February, 1984, Blum and Kahlert., 1978, Issell and Crooke , 1978, Chabner et al., 1978). Toxic effects prevented further development.

A class of agents that interfere with tubulin comprise taxanes (Payne 2003), especially highly potent ones and those containing thiol or disulfide groups. The taxanes are poisons of mitotic use that inhibit the depolymerization of tubulin, resulting in an increase in microtubule structure velocity and cell death. The taxanes which are within the scope of the present invention are for example described in US Patents. Numbers 6,436,931; 6,340,701; 6,706,708 and the Patent Publications of the U.S.A. Number 20040087649; 20040024049 and 20030004210. Other taxanes are described, for example, in U.S. Pat. Number 6,002,023, Patent of the U.S.A. Number 5,998,656, U.S. Patent No. 5,892., 063, U.S. Patent. No. 5,763,477, U.S. Pat. Number 5,705,508, Patent of the U.S.A. Number 5,703,247 and U.S. Pat. Number 5,367,086. As will be appreciated by the person skilled in the art, PEGylated taxanes such as those described in U.S. Pat. Number 6,596,757, they are also within the scope of the present invention.

The present invention includes additional effector molecules that affect DNA, more particularly interleaving agents such as anthracyclines and derivatives (daunorubicin, valrubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, amrubicin, pirarubicin, zorubicin) and anthracendins, such as substances derived from Streptomyces ( actinomycin, mitomycin, bleomycin, aactinomycin) or amsacrine.

An effector molecule can represent more particular DNA alkylating agents such as and more particularly nitrogen mustard and analogs (e.g., Cyclophosphamide, Melphalan, Estramustine), Alkylsulfonates, Nitrosoureas, Aziridines, Hydrazines, Ethylene Imines, and other substances such as Trenimon and Mitobronitol ( an analogue of mannitol). In particular, preferred DNA alkylating agents are analogs of CC-1065 or derivatives (U.S. Patent Nos. 5,475,092, 5,585, 499; 6, -716,821) and duocarmycin.

CC-1065 represents a potent antitumor antibiotic isolated from Streptomyces zelensis cultures and has been shown to be exceptionally cytotoxic in vitro (U.S. Patent No. 4,169,888). Within the scope of the present invention for example are the analogs CC-1065 or derivatives described in the patents of E.U.A. Numbers 5, 475,092, 5,585, 499 and 5,739,350. As the person skilled in the art will readily appreciate, analogs of CC-1065 or modified derivatives as described in U.S. Pat. Number 5,846,545 and prodrugs of analogues CC-1065 or derivatives as described, for example, in US Pat. Number 6,756,397, they are also within the scope of the present invention. In certain embodiments of the invention, analogs and derivatives of CC-1065 may for example be synthesized as described in US Pat. Number 6,534, 660.

Other DNA alkylation effector molecules such as platinum-based substances are also included (eg, carboplatin, nedaplatin, oxaliplatin, triplatine, satraplatin).

Effector molecules affecting DNA also include inhibitors of topoisomerase I and II such as substances derived from Camptotec (belotecan, topotecan) and podophyllotoxin and derivatives (etoposide, teniposide).

An additional subclass of effector molecules that affect DNA includes antimetabolites such as folic acid analogs (methotrexate, known as dihydrofolate reductase inhibitors) or Aminopterin. Metabolites that interfere with the metabolism of purine or pyrimidine, in particular adenosine deaminase inhibitor (pentostatin), or inhibitor of ribonucleotide reductase / halogenated (cladribine, clofarabine), thiopurine and thiazofurin are also included. Additional antimetabolite include DNA polymerase inhibitor (cytarabine), inhibitor of ribonucleotide reductase (gemcitabine), and hypomethylating agents (azacitidine, decitabine) and inhibitors of ribonucleotide reductase. More generally, DNA crosslinking substances such as cisplatin are also included.

An effector molecule according to the present invention can be antitumor antibiotics defined as effector molecules that damage or modify the DNA including enediin antibiotics such as calicheamicin including for example gamma II, N-acetyl calicheamicin and other calicheamicin derivatives. Calicheamicin binds in a specific sequence form with the minor groove of DNA, undergoes rearrangement and exposes free radicals, leading to double-stranded DNA breakdown, resulting in apoptosis and cell death. An example of a calicheamicin effector molecule that can be employed in the context of the present invention is described in U.S. Pat. Number 5,053,394. This compound is used in immunoconjugates with published monoclonal antibodies such as gemtuzumab, ozogamicin and irtotuzumab ozogamicin.

A subgroup of enediin comprises the cromoproteins esperamycin and neocarzinostatin. In particular, Trabectedin, which is also categorized as an agent that damages DNA, called antitumor antibiotics.

Trabectedin causes rupture of the main DNA structure, and can be isolated from an ascidian (also known as ecteinascidin 743 or ET-743) sold by ZELITA and JOHNSON & JOHNSON under the brand YONDELIS.

Another group of preferred effector molecules are substances such as but not limited to toxins that affect cell metabolism. In particular enzyme inhibitors such as but not limited to olaprib or more preferred proteasome (for example bortezomib) and protein kinase inhibitors or lipoxygenase inhibitors such as masoprocol are part of the present invention. Also included are receptor antagonists such as but not limited to endothelin A receptor antagonist (eg atrasentan), or sex steroids such as testolactone, which interfere with estrone metabolism. Also included are substances that interact with estrogen receptor such as polyphenols derived from plants, for example but not only isoflavonoids, stilbenes, silymarin, phenylpropanoid glycosides referred to as hitoestrogens.

Also suitable as effector molecules are substances that affect cellular metabolism, such as substances employed for photodynamic or radiation therapy including but not limited to porphyrin derivatives, for example d-Aminolevulinic acid. Efaproxiral represents a radiosensitizer, which increases oxygen levels by decreasing the affinity of oxygen-hemoglobin. They also include retinoids (first, second and third generation), in particular Tretinoin (ATRA), all trans retinoic acids that are used to treat acute promyelocytic leukemia (APML = Acute Promyelocytic Leukemia) sold for this indication by ROCHE under the brand name VESANOID. Retinoids are a class of chemical compounds that are chemically related to vitamin A, exerting diverse functions such as for example activation of tumor suppression genes. Currently, they are used to treat skin cancer and inflammatory skin disorders.

In another preferred embodiment, effector molecules can affect signaling pathways such as but not limited to calcium signaling. Examples are arsenic trioxide or trimethyltin chloride, the latter being a highly toxic organotin compound.

The present invention also includes effector molecules that affect drug resistance mechanisms that may include, for example, anti-drug multidrug resistance activity (by P-glycoprotein inhibition). Bicyclic heteroaromatic compounds and derivatives can serve as non-limiting examples.

Another class of effector molecule may include substances, or more particularly proteins that interfere with an apoptotic signaling pathway, including, but not limited to, antisense oligonucleotides, more particularly oligodeoxynucleotides such as Oblimersen (INN, Genasense brand, also known as Augmerosen and oligodeoxynucleotide). antisense bcl-2 G3139), which is an antisense oligodeoxyribonucleotide currently studied as a possible treatment for several types of cancer, including chronic lymphocytic leukemia, B-cell lymphoma and breast cancer. It has been proposed that this compound can kill cancer cells by blocking the production of Bcl-2 and by making them more sensitive to chemotherapy. A class of additional apoptosis inducing substances that can serve as effector molecules, comprises plant polyphenols such as, but not limited to, silymarins, which are capable of interfering with cell cycle regulators and proteins involved in apoptosis.

Other effector molecules may include enzymes such as but not limited to, asparaginase or other enzymes with antineoplastic activities.

A drug effector molecule according to the present invention may also be an antiprotozoal drug such as Miltefosine.

In another embodiment the effector molecules may represent plant polyphenols, such as but not limited to, psoralens and their hydroxy metabolites.

Plant polyphenols such as flavonoids, tannins (proanthocyanidins), stilbenoids, curcuminoids and lignans have one of the aforementioned antitumor activities (for example that induce apoptosis, cell cycle brake) or additional activity such as scavengers or free radical receptors, activity metal chelator, estrogen receptor interference activity, antioxidant, which interferes with enzymes that metabolize drugs) are also possible effector molecules. More specifically, psoralens and their hydroxy metabolites which are capable of intercalating in DNA that act as metal chelators having antioxidant and cytoprotective properties are preferred effector molecules. Particularly preferred are reservatol and polyhydroxy derivatives and flavonoids, such as catechins and epicatechins, more specifically epigallocatechins 3-0 gallate, which can act as antioxidants.

A broad classification of effector molecules according to their mechanism is also possible: Antineoplastic agents and immunomodulatory agents (in accordance with ATC code L01) in particular "Intracellular Chemotherapeutic Agents" ATC: Anatomical Therapeutic Chemical Classification System (WHO) 1) Antimycotics, or molecules that affect microtubules (tubulin binding agents) such as vinca alkaloids and analogues (Vinca alkaloids (Vinblastine, Vincristine, Vinflunine, Vindesine, Vinorelbine) and Taxans (Paclitaxel, Larotaxel, Docetaxel) dolastatins (and derivatives by example auristatin) and critoficin, maytansins and derivatives of colchicine, epothilones (for example ixabepilone) 2) that affect DNA replication a) Interlealing agents such as Ampheraciclins (Daunorubicin, Valrubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Zorubicin) and anthracendins, such as substances derived from Streptomycins (Actinomycin, Mitomycin, Bleomycin, Dactinomycin) or Amsacrine b) Alkylating agents such as nitrogen mustards, Nitrosoureas, Alkylsulfonates, Aziridines, Hydrazines (Procarbazine), Triazenes, Epoxides, Ethylene Imines, Altretamine, Mitobronitol, dupcarmycin and analogues / stereoisomers, Trenimon, Estramustine, CC-1065 c) Alkylating agents such as Platinum (for example Carboplatin, Nedaplatin, Oxaliplatin, Triplatin Tetranitrate, Satraplatin) d) Topoisomerase I specific inhibitors such as camptotheque (Belotecano, Topotecan) e) Topoisomerase II specific inhibitors such as podophyllotoxin and derivatives (Etoposide, Teniposide) f) Antimetabolites that affect the synthesis of DNA / RNA by interference with folic acid such as Dihydrofolate reductase inhibitors (eg, Aminopterin, Methotrexate), thymidylate synthase inhibitor Purine such as adenosine deaminase inhibitor (Pentostatin), ribonucleotide reductase / halogenated inhibitor (Cladribine, Clofarabine), Thiopurine, Thiazofurin - Pyrimidine such as DNA Polymerase inhibitor (Cytarabine), ribonucleotide reductase inhibitor (Gemcitabine), hypometalating agent (Azacitidine, Decitabine) - deoxyribonucleotide such as inhibitor of ribonucleotide reductase Hydroxycarbamide g) other DNA entanglement agents such as platinum-based compounds (e.g. Cisplatin) 3) Other substances that interfere with DNA eg "cytotoxic / antitumor antibiotics" such as Elsamicin A, additional antibiotics such as CC-1065, and subclasses of antibiotics such as calicheamicin enediin or derived from bacteria or chromium protein enedin, Esperamycin (combination agents of extremely toxic DNA) or Neocarzinostatin (Other members of the neocarzinostatin antibiotic group are macromycin, actinoxanthin, kedarcidin, and maturepeptin) or Trabectedin (breakdown of the DNA backbone) 4) toxins that affect the cellular metabolism for example inhibitors HSP90, Lonidamide (both inhibitors of respiration and glycolysis leading to a decrease in cellular ATP) a) Inhibitors of enzymes for example Olaprib (PARP inhibitor), CDK inhibitors (Alvocidib), · - Proteasome (Bortezomib), Protein kinase inhibitors, Masoprocol (Lipoxienase inhibitor) b) Receptor antagonists such as tutin (Glycine receptor antagonist (plant toxin), Atrasentan, retinoid X receptor (Bexarotene), sex steroids such as testolactone, substances that interfere with the estrogen receptor c) Photosensitizers or other compounds used for photo-dynamic therapy (Porfirmer Sodium), Porphyrin derivatives for example α-Aminolevulinic acid) d) Radiosensitizer such as Efaproxiral which increases oxygen levels by decreasing the affinity of hemoglobin-oxygen e) Substances that affect the signaling pathways for example Ca2 + signaling such as arsenic trioxide and trimethyltin chloride f) Other substances that interfere with metabolism such as retinoids and Tretinoin derivatives (ATRA) 5) Affecting epigenetic processes such as HDAC inhibitors (eg Panobinostat, Vorinostat, Valporic acid, MGCD0103 (Mocetinostat), which are currently in clinical development for cutaneous T-cell lymphoma, acute myeloid leukemia, Hodgkin's lymphoma or lymphoma follicular) 6) That affect drug resistance mechanisms such as bicyclic heteroaromatic compounds, which inhibit P-glycoprotein 7) Substances that induce apoptotic signaling mechanisms that include proteins but also antisense oligodeoxinucleotides such as Oblimersen (Genasense brand) 8) Enzymes such as Asparaginase 9) Antiprotozoal drugs such as Miltefosina 10) Plant polyphenols such as Flavonoids, Tannins (Proanthocyanidins), Stilbenoids, curcuminoids and lignans that have one of the aforementioned antitumor activities (for example that induce apoptosis), cell cycle brake) or additional activity such as scavenging or free radical scavenging, metal chelating activity, estrogen receptor interference activity, antioxidant, which interferes with enzymes that metabolize drugs). More specifically, soralenes and their hydroxy metabolites, ol reserve and polyhydroxy derivatives, Flavonoids, such as Catechizas and Epicatequinas, more specifically epigallocatechins 3-0 gallate 11) Additional natural substances and derivatives such as Eotoxin A, Diphtheria toxin and its derivatives, where the derivatives can be modified in chemical or genetic form.

Effector molecules can also be categorized according to the class of substance to which they belong such as inorganic compounds, aromatics, metal-based compounds, proteins related to cellular metabolism, enzymes, peptides, oligonucleotides, such as antisense nucleotides, bacterial toxins, toxins derived from plants and polyphenols such as tannins, flavonoids and coumarins as well as terpenoids, alkaloids, anti-tumor antibiotics (for example, enediin antibiotics), mycotoxins, invertebrate toxins as well as vertebrates, environmental toxins.

An immunoconjugate according to the present invention comprises at least one targeting agent, in particular targeting antibody and an effector molecule. The immunoconjugate may comprise additional molecules for example for stabilization. For immunoconjugates, the term "conjugate" is generally used to define the operative association of the targeted agent with one or more effector molecules and is not intended to refer only to any type of operative association and is not particularly limited to "conjugation" chemistry. As long as the targeted agent is capable of binding to the target site and the aggregate effector works sufficiently as intended, particularly when the target site is supplied, any connection mode will be suitable. The conjugation methods according to the present invention include but are not limited to, direct connection of the effector molecule to the targeted antibody with or without prior modification of the effector molecule and / or the targeted antibody or in connection by linkers. The linkers can be functionally categorized, for example, in labile acids, photolabile linkers, cleavage linkers by enzymes, such as linkers that can be decomposed by peptidases. Binds susceptible to disruption are preferred in many embodiments of the invention. These linkers susceptible to disruption can be decomposed under conditions present in the cell environment, in particular an intracellular environment and which have no deleterious effect on the drug released upon decomposition. Low pHs such as pH of 4 to 5, as they exist in certain intracellular departments, will break labile acid linkers, whereas photolabile linkers can be broken down by infrared light for example. However, linkers that are broken by / under physiological conditions present in most cells are preferred and are referred to herein as physiological decomposition binders. Accordingly, disulfide linkers are preferred in many embodiments of the invention. These linkers are susceptible to disruption through disulfide exchange, which can occur under physiological conditions. Heterobifunctional disulfide linkers include, but are not limited to, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (see, eg, Carlsson et al. (1978)), N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) (see, for example, U.S. Patent Number 4,563, 304), N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) (see, eg, CAS Registry number 341498-08-6), N -succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (see, for example, Yoshitake et al., (1979)) and N-succinimidyl 4-methyl-4- [2- (5-nitro) pyridyl) -dithio] pentanoate (SMNP) (see, for example, U.S. Patent Number 4,563,304). The most preferred linker molecules for use in the composition of the invention are SPP, SMCC and SPDB.

Other suitable linkers may include "non-cleavable" linkages, such as but not limited to Sulfosuccinimidyl maleimidomethyl cyclohexane carboxylate (SMCC), which is a heterobifunctional linker capable of binding compounds with compounds containing SH. Bifunctional and heterobifunctional linker molecules, such as heterobifunctional linker molecules directed to carbohydrates, such as S- (2-thiopyridyl) -L-cysteine hydrazide (TPCH), are also within the scope of the present invention (Vogel, 2004). The effector molecule, such as a maitaisinoid, which can be conjugated to the targeted antibody by a two-step reaction process, including as a first step modification of the targeted antibody with an interlacing reagent such as N-succinimidyl pyridyldithiopropionate (SPDP) to introduce dithiopyridyl groups in the directed antibody. In a second step, a reactive maytansinoid having a thiol group, such as DM1, may be added to the modified antibody, resulting in the displacement of the thiopyridyl groups in the modified antibody, and the production of disulfide-linked antibody / cytotoxic maitaisinoid conjugate ( U.S. Patent No. 5,208,020). However, one-step conjugation processes such as that described in U.S. Patent Publication. No. 20030055226 to Chari et al., are also within the scope of the present invention. In one embodiment of the present invention multiple effector molecules of the same or different type are connected or linked to a targeted antibody. As discussed elsewhere, the nature of the linkers used can influence extermination by non-specific activity (Kovtun et al., 2006). See also the discussion of Figure 13. See also U.S. Patents. Numbers 5,208,030; 5,416,064; 6,333,410; 6,441,163; 6,716,821; 6,913,748; 7,276,497 and the U.S. Patent Application. Number 2005/0169933, method for preparing immunoconjugates.

Analogs CC-1065 or derivatives can be conjugated to the targeted agent by for example PEG linking groups as described in US Pat. Number 6,716, 821.

Calicheamicins can be conjugated to antibodies directed by linkers (U.S. Patent Number 5,877,296 and U.S. Patent Number 5,773,001) or according to the conjugation methods described in U.S. Pat. Number 5,712,374 and the U.S. Patent. Number 5,714,586. Another preferred method for preparing calicheamicin conjugates is described in U.S. Patent Publication. Number 20040082764. The immunoconjugates of the present invention may take the form of recombinant fusion proteins.

An operational association in the form of connection with or without a linker is referred to herein as "connection or functional link".

An immunoconjugate that essentially consists of certain components means in the context of the present invention that the antibody / immunoconjugate consists of the specified components and any additional materials or components that do not materially affect the basic characteristics of the antibody.

Figure 9 shows in (C) and (D) the differences in link / direction homogeneity between immunoconjugates comprising murine antibody BB4 (BB4-SPP-D 1; Figure 9C) and the engineered engineered antibody nBT062 (nBT062-SPP -DM1; Figure 9D) based on it. As can be seen from these graphs, the results obtained with the immunoconjugate comprising the engineered engineered antibody are substantially more homogeneous than those obtained with the immunoconjugates comprising the murine antibody. This is particularly remarkable since the antibody binding region of BB4 was not modified in nBT062. In this manner, the immunoconjugate comprising the antibody binding region of the murine antibody, but not other parts of the murine antibody, showed properties that far exceed the results that would have been expected by the person skilled in the art.

Some of the immunoconjugates of the present invention have an effector molecule that is sterically hindered, and contains a segmentable linker, hindered immunoconjugate, HICL-Hindered Immunconjugate, Cleavable Linker. An unimpeded counterpart (UI: unimpeded immunoconjugate (UI = ünhindered Immunoconjugate) of an immunoconjugate comprising an engineered antibody against CD138 linked to an effector molecule by a segmentable linker (CL) and described herein as UICL. an immunoconjugate equivalent to HICL comprising an engineered engineered antibody wherein the effector molecule is however not sterically hindered.Examples of a pair of HICL / UICL are BT062 and nBT062-SPP-DM1 An unimpeded counterpart of this immunoconjugate comprising a non-cleavable linker (UINCL) refers to the equivalent immunoconjugate comprising an engineered antibody engineered in which the effector molecule is not sterically hindered and comprises a non-segmentable linker For BT062 (nBT062-SPDB-DM4), nBT062-SMCC- DMl will constitute an example of this unimpeded counterpart comprising a non-segmentable linker (UNICL).

A growth of a tumor inhibitory activity (= tumor growth inhibitory activity) of an immunoconjugate is a relative measure. Describes the inhibitory activity of tumor growth of a conjugate with respect to the activity of the highest performance immunoconjugate whose activity is established as 100%. For example, if the activity of the highest performance immunoconjugate, say BT062 will cause a delay in tumor growth. (TGD = Tumor Growth Delay) of 32 days, is adjusted as 100%, the activity for example of nBT062-D l that exhibits a tumor growth delay (PDD) of 18 days is calculated as follows: Inhibitory Activity of Tumor Growth lOOx (TGDnBT062-D i / TGDBT062), in a more generic way: Inhibitory Activity of Tumor Growth = lOOx (TGDMuestra / TGDReferencia) · Table 6 provides convenient examples from the results illustrated in Figure 11B: TGD * (days)% Activity ** PBS 0 0 nBT062-SMCC-DMl 18 56 BT062 32 100 nBT062-SPP-DMl 13 40 Table 6: Tumor growth delay (TGD) and Activity% of nBT062-DMx against MOLP-8 tumor xenografts in SCID mice based on the treatment groups receiving a dose of 450 g / kg.

(*) Delay of growth - of tumor in days (TGD) as average time in days for the treatment group to reach a predetermined size (160 mm3) less the average time for the control group to reach this predetermined size.

(**) Tumor Growth Inhibitory Activity = 100x (TGDMUeStra / TGDBT062) · BT062 activity is defined as 100%.

In the example provided in Table 6, BT062 provides a growth of a tumor inhibitory activity that exceeds that of its unimpeded counterpart (nBT062-SPP-DMl) by 60%, and a growth of tumor inhibitory activity that exceeds that of its unimpeded counterpart immunoconjugate comprising a non-segmentable linker (nBT062-SMCC-DMl) in 44%.

As discussed above, certain drugs such as maytansinoids, while effective, are highly toxic, destroying cells in their non-conjugated, native form, nonselectively. The binding of the cytotoxic maytansinoid to an antibody can keep the drug inactive until it reaches the target cell (Lambert 2005). Several conjugates antibodies-maytansinoid are under clinical development.

Phase I and II studies were performed with IMGN901 (huN901-DMl, BB-10901) to treat CD56-positive solid tumors (small cell lung cancer and neuroendocrine cancers). In these studies I GN901 was administered in 4 consecutive weeks every 6 weeks, and in general it was well tolerated (Fossella et al., 2005, Lorigan et al., 2006, McCann et al., 2007, Carter and Senter, 2008, Johnson et al., 2008). The antibody portion of the huN901 immunoconjugate shows significant CDC or ADCC activity. The same immunoconjugate is investigated for CD56 positive multiple myeloma treatment. In a Phase I study, administration of IMGN901 in 2 consecutive weeks every 3 weeks to a patient with CD56 positive multiple myeloma who has failed established multiple myeloma treatments, has shown preliminary safety evidence as well as clinical activity. Ten and eight patients were reported to have received IMGN901 (3 patients each at 40, 60, 75, 90, 112, and 140 mg / m2 / week). Preliminary PK results were reported indicating, an approximately linear relationship between dose and maximum serum concentration observed. Interesting clinical activity has been observed with a tolerable safety profile. A small (MR = Minor Responce) confirmed response was documented in "3 heavily pretreated patients (1 patient each with 60, 90, and 112 mg / m2 / week) using the European criteria for Bone Marrow Transplantation. Durable at doses of 60, 90, 112, and 140 mg / m2 / week (Chanan-Khan et al., 2007, Chanan-Khan et al., 2008) IMGN901 is also investigated in a Phase I study in tumors The immunoconjugate is administered daily for 3 days every 3 weeks.Preliminary clinical activity has been noted in patients with small cell lung cancer, merkel cell carcinoma and other solid tumors.The dose scale continues.

MLN2704 (huJ591-DMl) is investigated to treat castration-resistant prostate cancer (Milowsky et al., 2006, Brand and Tolcher 2006). A Phase I trial of MLN2704 in patients with progressive metastatic castration-resistant prostate cancer investigates the safety profile, pharmacokinetics, immunogenicity, and antitumor activity of MLN2704 when administered once every four weeks. Results show that therapeutic doses of MLN2704 can be administered safely on a repeatable basis (Galsky et al., 2008). Parallel tests were performed with another immunoconjugate-DM1, that is, bivatuzumab mertansin that targets Cd44v6, which is expressed in carcinomas of the head and neck and other solid tumors. In the clinical trial with the most condensed administration program (weekly administration) binding to CD44v6 in skin keratinocytes mean serious toxicity in the skin with a fatal outcome in a patient, which led to termination of the bivatuzumab mertansine development program (Tijink et al. al., 2006, Sauter et al., 2007, Rupp et al., 2007, Riechelmann et al., 2008).

CD44v6 is not only expressed in various cancer cells, but also in normal skin tissue and resembles in this aspect CD138 which is also expressed not only in cancer cells but in normal skin tissue. Surprisingly, it was found that BT062 shows clinical efficacy without intolerable side effects such as skin toxicity as found in bivatuzumab mertansine. See Figure 23, which shows that repeated single doses of BT062 of up to 160 mg / m2 lead to at least stable disease with manageable side effects. The results illustrated in Figure 23 also show that 10 repeated single doses of 20 mg / m2 (treatment over 6 months), 5 repeated single doses of 40 mg / m2, 5 repeated single doses of 80 mg / m2, 6 doses repeated single doses of 160 mg / m2, and a single single dose of 200 mg / m2, followed by 6 repeated single doses of 160 mg / m2 (ergo, a total dose of 1160 mg / m2) were well tolerated (patients associated with 003 -005 and 002-012, and 002-011 are still in the process of being processed).

CD138 is also expressed in normal blood cells and other cells, such as epithelial cells, whose destruction would lead to intolerable side effects. Regardless of this, no dose-limiting toxicity was found to non-cancer / non-tumor cells expressing CD138 of any type in the treatment regimens illustrated in Figure 23 to 120 mg / m2, while the maximum tolerable dose (MTD = Maximum Tolerable Dose) was determined in this study as 160 mg / m2 and the maximum administered dose (MAD = Maximum Administered Dose) was determined to be 200 mg / m2. Doses between 160 mg / m2 and 200 mg / m2 were not tested. Higher doses were generally not given in view of dose limiting toxicities (DLT = Dose Limiting Toxicities) at 200 mg / m2. However, it was observed that the dose of DLT was still acceptable when administered with subsequent minor doses such as 160 mg / m2 dose. A clinically non-significant toxicity against non-cancer / non-tumor cells expressing CD138 (non-target cells expressing CD138) is referred to herein as "clinically acceptable toxicity" or "tolerable toxicity" against said cells and the respective organ (s). The respective amount of immunoconjugate that is administered is referred to herein as "tolerable amount". Thus, in one embodiment of the present invention, the immunoconjugate exhibits clinically acceptable toxicity to these non-target cells expressing CD138, in particular epithelial cells expressing CD138, which are not detected by BT062 in the mouse xenograft model because to human specificity.

Clinically acceptable toxicity includes the performance of an adverse reaction, to a manageable or tolerable level. An adverse reaction is defined as an undesirable effect, reasonably associated with the use of a drug, here the immunoconjugate, which may occur as part of the pharmacological action of the drug or may not be predictable in its occurrence. This definition does not include all the adverse events observed during the use of a drug, only those for which there is some basis to believe that there is a causal relationship between the drug and the occurrence of the adverse event. Adverse reactions - may include signs and symptoms, changes in laboratory parameters and changes in other measures of critical body function, such as vital signs and ECG.

If a cell, such as a cell does not target especially a non-target cell (non-tumor cell) expressing CD138, is said to be "substantially unaffected" by the administration, in particular the administration of a certain dose of a compound such as a immunoconjugate, means that any interaction that this compound has had with the non-target cell resulted in any or a tolerable / manageable adverse reaction.

Phase I studies with the immunoconjugate form of 'trastuzumab (T-DM1) for treatment of metastatic breast cancer that overexpresses HER2 are performed to investigate safety and pharmacokinetics of T-DM1 administered weekly once every 3 weeks. In both studies AEs of degree >; 2 related to T-DMl has been infrequent and manageable. Objective tumor responses have been observed at doses at or below BAT (Burris et al., 2006, Krop et al., 2007, Beeram et al., 2008, Holden et al., 2008). A Phase II study investigating T-DMl in HER2 positive metastatic breast cancer when administered once every 3 weeks has been initiated (Beeram et al., 2008, Carter and Senter, 2008, Holden et al., 2008). A Phase III clinical trial evaluating T-DM1 for HER2-positive second-line metastatic breast cancer and Phase II clinical trials evaluating T-DM1 for metastatic breast cancer HER2-positive first, second and third lines are in process. A Phase Ib clinical trial in combination with pertuzumab for HER2-positive metastatic breast cancer patients who have progressed on treatment based on Herceptin is planned. Three phase I clinical trials have been completed with cantuzumab mertansine, a DM1 conjugate of the huC242 antibody that targets or targets an antigen found in colorectal cancers and other cancers that express C242. Treatment with huC242-DMl administered on a weekly basis as well as once every 3 weeks was found to be safe and tolerated (Rowinsky et al., 2002, Tolcher et al., 2003, Helft et al., 2004).

Four studies investigate immunoconjugates using the thiol-containing maytansinoid DM4, which is also a BT062 component: An analog of cantuzumab mertansine, IMGN242 (huC242-DM4), was investigated in a phase I study in cancer subjects expressing CanAg (Tolcher et al., 2006). Subjects received a single IV infusion of IMGN242 once every 3 weeks with a dose in the range of 18 to 297 mg / m2. Dose limiting toxicity was experienced by 2 of 6 subjects treated at the 223 mg / m2 dose level during their second treatment cycle. The drug was well tolerated at the 168 mg / m2 level and does not induce any detectable antibody response (Mita et al., 2007). Based on the first safety results of the Phase I study, a Phase II study was started to evaluate IMGN242 to treat gastric cancer that expresses CanAg for doses of 168 mg / m2 (Sankhala et al., 2007). Forty-five patients have been treated with IMGN242 in two clinical trials. Based on safety and complete pharmacokinetic (PK) / pharmacodynamic (PD) clinical analyzes, the Phase II study was amended to treat patients with low levels of CanAg in plasma at a dose of 126 mg / m2 and patients with high levels of CanAg. in plasma at 168 mg / m2 (Qin et al., 2008).

A phase I study with huMy9-6 antibody conjugated to DM4 (AVE9633) was also performed for the treatment of subjects with CD33-positive acute myeloid leukemia (AML). The treatment regimen consisted of IV infusions once every 3 weeks using a dose range of 15 to 260 mg / m2. Neither associated myelosuppression nor responses have been noted in a single-dose study (Giles et al., 2006). A second phase I study investigating AVE9633 with the treatment regimen consisting of IV infusions on day 1 and day 8 of a 28-day cycle also showed that AVE9633 was well tolerated and shows evidence of antileukemia activity including a subject with a response complete (inadequate platelet response, transfusion-dependent) that lasts at least 4 months (Legrand et al., 2007). Two additional immunoconjugates DM4 (SAR3419 and BIIB015) have entered Phase I clinical trials.

Also, it is known from other immunoconjugates such as Mylotarg that target or target CD33, that the activity of the immunoconjugate may not be sufficient to treat patients at low doses. This problem has been alleviated for example by administration of colony stimulus factor of recombinant human granulocytes (rhG-CSF = recombinant human Granulocyte Colony Stimulating Factor) to sensitize target cells expressing CD33 (Fianchi et al., Annals of Oncology 2008 19 (1): 128-134).

Previous studies demonstrate that responses to different immunoconjugates, in particular immunoconjugates containing maytansinoid (such as DM1 or DM4), vary widely. The BT062 tests in human subjects showed tolerable toxicity against non-cancerous cells expressing CD138 at different doses of stable disease, especially at doses of up to 160 mg / m2.

The immunoconjugate described herein can be administered in combination with cytotoxic agents. These combinations are referred to herein as anticancer combinations.

Currently, many combinations of anti-myeloma drugs in particular are investigated in clinical trials. The purpose of using a combination in general either to improve the effectiveness, to overcome a refractory phenotype, for example, of myeloma cells, to reduce side effects due to the use of lower concentrations of one of the combination partners or a combination thereof. It was shown to reduce toxicity using a low dose, for example, of lenalidomide plus a low dose of dexamethasone (Rajkumar et al., 2010).

Especially in patients with relapsed or refractory multiple myeloma, several combinations of drugs have been and have been investigated.

A standard example for combining chemotherapeutic agents represents the triple combination of vincristine, dexamethasone, doxorubicin (VAD regimen).

Proteasomal inhibitors such as bortezomib have been combined with myeloma drugs, such as melphalan and prednisone (VMP). This combination resulted in a complete response rate of 16% and a total response rate of 89% (Mateos et al., 2006).

Bortezomib has also been approved for use in combination with liposomal doxorubicin for relapsed or refractory patients (Ning et al., 2007).

Bortezomib is investigated in several clinical studies for use in combination with dexamethasone, melphalan, prednisone and / or thalidomide.

Bortezomib is also under investigation, combined with liposomal doxorubicin, cyclophosphamide and dexamethasone in multiple myeloma patients. Combinations with Vorinostat are currently under investigation aimed at resensitizing patients to bortezomib who are refractory to this drug.

Thalidomide, which is administered orally, has been combined with melphalan / prednisone (MPT) (Facón et al., 2006) or dexamethasone or bendamustine (Ponisch et al., 2008).

Furthermore, lenalidomide, an immunomodulatory drug, used in combination with dexamethasone, resulted in a prolonged time to tumor progression and increased survival compared to dexamethasone alone (Weber et al., 2006). Lenalidomide combined with dexamethasone has also been studied in recently diagnosed patients (Rajkumar et al., 2005) as well as the combination with melphalan / prednisone (RMP) (Palumbo et al., 2006).

The Patent Publication of the E.ii.A. No. 2010/0028346 issued to Lutz et al., Describes synergistic effects of certain immunoconjugates with chemotherapeutic agents.

In the present context, a goal of employing combinations is a reduction in the effective dose of the immunoconjugate of the present invention, reducing its side effects and opening new therapeutic windows with acceptable side effects. Another goal is to reduce the effective dose of previously used cytotoxic agents such as VELCADE or lenalidomide and preferably reduce the side effects of these agents. Similarly, the positive consequences of doses include, but are not limited to, prolongation of treatment, higher doses, other application programs, better and more sustained response to treatment.

Patients exhibiting a refractory phenotype to drugs such as lenalidomide, melphalan (study in progress) can be made sensitive again by the use of immunoconjugates according to the present invention.

The term "cytotoxic agent" comprises "cytotoxic / cancer drugs" including chemotherapeutic agents, in particular chemotherapeutic agents which, in general, are used in rapidly dividing cells, ie: Alkylating agents such as nitrogen mustards (for example melphalan, cyclophosphamide, mechlorethamine, uramustine, chlorambucil, ifosfamide) or nitrosureas (e.g. carmustine, lomustine, streptozocin) or alkylsulfonates; - Alkylating agents such as cisplatin, carboplatin, nedaplatin, oxaliplatin; or non-classical alkylating agents such as tetrazines, dacarbizine, procarbazine, altretamine Anthracyclines such as doxorubicin and liposomal doxorubicin (DOXIL) - Alkaloids such as vincristine The term "cytotoxic agents" also comprises immunomodulatory drugs (ImiDs = imunomodulatory drugs) such as thalidomide (or analogues), lenalidomide (CC-5013), pomalidomide, actimid, which are used for myeloma therapy in view of their pleiotropic immunomodulatory properties. They commonly exhibit anti-inflammatory activity by inhibiting TNF alpha production, but also exhibit anti-angiogenic activity and immunomodulatory properties such as co-stimulation of T cells and influence on regulatory T cells (Quach et al., 2010).

The term "cytotoxic agent" also comprises steroids, such as, but not limited to, dexamethasone and prednisone as well as proteosomal inhibitors such as bortezomib (VELCADE) or carfilzomib which induce the activation of programmed cell death in neoplastic cells dependent on path suppression. -Apoptotic Additional cytotoxic agents. potent, include etoposide, which inhibit the enzyme topoisomerase II, cytarabine, which before conversion damages DNA when a cell cycle is maintained in S phase (DNA synthesis) and in this way in particular affects rapidly dividing cells such as cancer cells . In addition, microtubule inhibiting agents such as vinca alkaloids, taxanes (as described above in the context of effector molecules) can also serve as cytotoxic agents according to the present invention.

Also s, and includes in the definition kinase inhibitor such as inhibitors sorafenib or HDAC (histone deacetylase) such as romidepsin as well as growth inhibitory agents, anti-hormonal agents, anti-angiogenic agents, cardioprotective, immunostimulatory agents, immunosuppressive agents, inhibitors of angiogenesis, protein tyrosine kinase inhibitors (PTK = Protein Tyrosine Kinase).

Also included in this definition are antibody-based cytotoxic agents including immunoconjugates and antibodies that have a recognized cytotoxic effect in the art. Anti-CD40 is a preferred antibody. Other antibodies include, but are not limited to, for example AVASTIN (bevacizumab) or MYELOMACIDE (milatuzumab).

Talomide (a- (N-phthalimido) glutarimide; thalidomide), is an immunomodulatory agent. The empirical formula for thalidomide is Ci3Hy0N2O4 and the molecular weight in grams is 258.2. The CAS number of thalidomide is 50-35-1. It seems to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells in various ways and to inhibit the growth of new blood vessels.

Lenalidomide (REVLIMID) is a derivative of thalidomide that represents the second generation of immunomodulatory compounds (ImiDs = immunomodulatory compounds) that initially developed as inhibitors of TNF alpha. Effects of lenalidomide include growth arrest or apoptosis, abrogated adhesion of myeloma cells to bone marrow stromal cells and modulation of cytokines that promote cell growth, survival and drug resistance of myeloma cells (Morgan et al., 2006). Lenalidomide is effective in patients refractory to thalidomide. In addition to effects on immune cells, ImiDs such as lenalidomide were suggested to cause cell cycle brake in G0 / G1 phase. In addition, it is considered that ImiDs down regulate the receptors of cleular adhesion (VLA-4, VLA-5, CD138) (Quach et al., 2010).

A down regulation of CD138 will be expected to cause a reduced binding of any agent that targets CD138, such as BT062, to dia cells.

Proteasome inhibitors can be divided into additional subgroups: a) peptide derivatives of natural origin having a C-terminal epoxy ketone structure, beta-lactone derivatives, aclacinomycin A, lactacystin, clastolactacystin; Y b) synthetic inhibitors (comprising modified peptide aldehydes, alpha structures, beta epoxyetone, vinyl sulfones, boric acid residues, pinacholesters.) A preferred proteasomal inhibitor of the present invention is bortezomib (PS 341; VELCADE, see discussion below). One of the proposed mechanisms suggests that proteasomal inhibition may prevent degradation of pro-apoptotic factors, allowing activation of programmed cell death in neoplastic cells dependent on suppression of pro-apoptotic pathways.In addition, bortezomib causes G2 / M cell cycle brake (Wang et al., 2009) Thus, bortezomib can interfere with anti-mitotic agents that are part of the immunoconjugate of the present invention, for example with the effect of maytansinoid DM4, which also acts in this phase of the cell cycle. The segmentation of PARP (Poly (ADP-ribose) Polymerase), which is carried out in apoptosis, is also affected so by DM4 as bortezomib. Accordingly, the combination of an immunoconjugate comprising an anti-mitotic agent, and a proteasomal inhibitor exhibiting the characteristics of bortezomib, is not adapted to the general guidelines previously established to obtain synergistic effects (Takimoto et al, 2009). .

VELCADE (bortezombid) is a proteasome inhibitor used to treat multiple myeloma. It is considered that VELCADE acts on myeloma cells to cause cell death, and / or acts indirectly to inhibit myeloma cell growth and survival by action in the bone microenvironment. Without being limited to a specific theory or mode of action, VELCADE in this way interrupts normal cellular processes, resulting in proteasome inhibition that promotes apoptosis.

Dexamethasone is a synthetic glucocorticoid steroid hormone that acts as an anti-inflammatory and immunosuppressant. When given to cancer patients, dexamethasone can counteract the side effects of cancer therapy. Dexamethasone can also be administered alone or together with other anticancer agents, including thalidomide, lelinalidomide, bortezomib, adriamycin or vincristine.

Substances for treatment, which may be used in combination with BT062 also include immunomodulatory agents (for example thalidomide and lenalidomide, and pomalidomide), proteasome inhibitors (for example bortezomib and carfilzomib), steroids (for example dexamethasone), alkylating agents and high-dose chemotherapy. doses, combinations (for example Melphalan and Prednisone (MP), Vincristine, doxorubicin (Adriamycin), and dexamethasone (VAD)), and bisphosphonates.

The expression "in combination with" is not limited to administration at exactly the same time. For him i otherwise, the term encompasses administration of the immunoconjugate of the present invention and the other regimen (for example radiotherapy) or agent, in particular the cytotoxic agents previously referred to in a sequence and within such a period of time that they can act in concert to provide a benefit (eg, increased activity, decreased side effects) that is increased compared to treatment with only any immunoconjugate of the present invention, or for example the other agent (s). It is preferred that the immunoconjugate and the other agent or agents act additively, and especially it is preferred that they act synergistically. These molecules are conveniently provided in amounts that are effective for the intended purpose. The medical practitioner with skill in the art can empirically determine, or consider the pharmacokinetics and modes of action of the agents, the appropriate dose (s) of each therapeutic agent, as well as the appropriate timing and administration methods. As used in the context of the present invention "concurrent administration" refers to the administration at the same time as the immunoconjugate, often in a combined dosage form.

Synergistic effects, which are the effects of two components such .. as an immunoconjugate and a cytotoxic agent, which exceeds a strictly additive effect. These synergistic effects can be counter-attacked by a number of factors that are discussed further below.

Synergism has been calculated as follows (Yu et al., 2001, Gunaratnam et al., 2009): PROPORTION (r) = Expected FTV (combination) / FTV observed (combination) FTV: Fractional tumor volume (Fractional tumor volume) = average tumor volume (test) / average tumor volume (control) A relationship > 1 is considered as synergistic, while r < 1 is less than additive.

The relation (r) is, when it is over 1, it is also referred to as "PROPORTION OF SYNERGY".

The ACTIVITY RATING is another measure for the effects of a combination. This rating is based on the Logio cell extermination Cell Extermination Logio = (T-C) / Td x 3.32 wherein (T-C) or delay in tumor growth is the average time in days required for the tumors of the treatment group (T) and the control group (C) to reach a predetermined size (600 mm3). Td is the time of tumor duplication, based on the average tumor volume in the control mouse, and 3.32 is the number of cellular duplications per log of cell growth. (Bissery et al., 1991). A Logio cell extermination higher than 2.8 indicates that the combination is highly active, a logio cell extermination of 2.0-2.8 indicates that the combination is very active, a logio cell extermination of 1.3-1.9 indicates that the combination is active, a cellular extermination logio of 0.7-1.2 indicates that the combination is moderately active and a logio cell extermination less than 0.7 indicates that the combination is inactive.

Selection of drug combination partners A set of guidelines for the design of combination chemotherapy regimens has been developed (Takimoto, 2006). Complying with these guidelines in general will increase the chances that a particular combination achieves at least one of the three most important theoretical advantages of combination chemotherapy over single-agent therapy: 1) Maximize cell killing while minimizing toxicities and hosts by using agents with dose-limiting toxicities without interference; 2) Increase the range of drug activity against tumor cells with endogenous resistance to specific types of therapy; Y 3) Avoid or stop the development of new resistant tumor cells.

The recommended principles to consider for selecting agents for use in combination chemotherapy regimens include: a) select drugs that are known to induce complete remission as simple agents, b) select drugs with different modes of action and with additive or synergistic cytotoxic effects that should be combined, c) select drugs with different dose-limiting toxicities, d) select drugs with different resistance patterns to minimize cross resistance.

Also, drugs should be administered at their optimal dose and schedule (e), and administration should be made at consistent intervals, while the treatment-free period should be as short as possible to allow recovery of normal tissue (f) (Takimoto et al. al, 2009). * Synergistic effects or effects only additives can be counter-attacked by a variety of factors: For example, the components of an anti-cancer combination can inactivate each other, for example by linking together. In addition, a component of an anti-cancer combination may interfere with the mode of action of another component. For example: Lenalidomide down-regulates cell adhesion receptors such as CD138, which is the target or target of the immunoconjugate of the present invention (Quach et al., 2010). The proteasome inhibitor bortezomib causes G2 / M cell cycle brake (Wang et al., 2009) which is also affected by anti-mitotic agents. In this way, if the effector molecule of the immunoconjugate is a maytansinoid, it will share a target or target for action with bortezombid, which is considered disadvantageous.

Doses, routes of administration and recommended use of the cytotoxic agents according to the present invention that have been widely used in cancer therapy are known in the art and have been described in literature such as the Reference Manual for Physicians (PDR = Physician's Desk Reference). The PDR describes doses of agents that have been used in treatment of various cancers. The dosage and dose regimen of these cytotoxic agents that are effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician with skill in the art and can be determined by the physician. The 2006 edition of the (PDR) describes the mechanism of action and preferred dose of treatment and dose schedules for thalidomide (p 979-983), VELCADE (p 2102-2106) and melphalan (p 976-979). A person skilled in the art can review the PDR, using one or more of the following parameters, to determine dose and dosage regimen of the chemotherapeutic and conjugated agents that can be employed in accordance with the teachings of this invention. These parameters include: 1. full index according to a) Manufacturer b) Products (by company or brand name drug) c) category index (for example, "proteasome inhibitors", "DNA alkylating agents", "melphalan" etc.) d ) generic / chemical index (name of common drugs without brand). 2. Medicine color images 3. Product information, consistent with FDA labeling including a) Chemical information b) Function / action c) Indications and counter-indications d) Test research, side effects, warnings.

As will be appreciated by the person skilled in the art, the amino acid sequence of the engineered engineered antibody portion of an immunoconjugate, nBT062, can be varied without loss of functionality of the antibody portion directed to CD138. This is particularly true when the heavy chain variable region CDR3 comprising amino acid residues 99 to 111 of SEQ ID NO: 1, and the CDR3 light chain variable region comprising amino acid residues 89 to 97 of SEQ ID NO: 2, respectively of the antigen binding region (ABR = Antigen Binding Region). Advantageously, the heavy chain variable regions CDR1 and CDR2 comprising amino acid residues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and / or (b) light chain variable region CDR1 and CDR2 comprising amino acid residues 24 a 34 and 50 to 56 of SEQ ID NO: 2, respectively of the antigen binding region (ABR) are also maintained.

The term "sequence identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned in such a way that the highest order correspondence is obtained. "Identity", per se, has recognized significance in the art and can be calculated using published techniques. (See, eg, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are a number of methods for measuring identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to people with dexterity (Carillo, H. &Lipton, D., SIAM J Applied Math 48: 1073 ( 1988)).

If any particular nucleic acid molecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, for example to the nucleic acid sequence nBT062, or a part thereof, each can be determined in conventional manner using known computer programs such as the DNAsis program (Hitachi Software, San Bruno, Calif.) for initial sequence alignment followed by ESEE version 3.0 DNA / protein sequence software (cabot@trog.mbb.sfu.ca) for multi-sequence alignments.

If the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, for example to SEQ ID N0: 1 or SEQ ID NO: 2, or a portion thereof, can be determined in conventional manner using well-known computer programs such as the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park , 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best homology segment between two sequences.

When DNAsis, ESEE, BESTFIT or any other sequence alignment program is used to determine if a particular sequence for example is 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage Identity is calculated over the entire length of the reference nucleic acid or amino acid sequence and spaces in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

If, in the context of the present invention, reference is made to a certain sequence identity with a combination of residues of a particular sequence, this sequence identity refers to the sum of all the specified residues.

As discussed above, BT062 is an immunoconjugate comprising the chimeric antibody directed to CD138 having nBT062 which is linked via a linker, here SPDB, to the cytostatic cytostatic derivative DM4. A chemical representation of BT062 is provided in Figures 1 and 2. Immunoconjugates comprising nBT062 and an maytansinoid effector molecule are often characterized in terms of their maytansinoid linker and effector, eg, nBT062-SMCC-DMl, is an immunoconjugate comprising nBT062, SMCC (a "non-segmentable" linker containing a thioester linkage) and DM1 as an effector. More generally, an immunoconjugate containing nBT062 and an effector molecule can also be described as an effector-linker-nBT062 or only as an effector nBT062 (nBT062N, where N is any effector described herein (see also Patent Publication of USA 20090232810).

In one embodiment, BT062 binds multiple positive myeloma cells to CD138. Once the target cell internalizes and / or releases immunoconjugate, DM4 is released from the targeted molecule, thereby restoring its original cytotoxic potency to DM4. In this manner, BT062 provides a targeted antibody payload (??? = Targeted Antibody Payload), wherein the functional binding of DM4 to nBT062 keeps the cytotoxic drug inactive until it reaches / internalizes into the directed cell expressing CD138.

Data from non-clinical studies investigating BT062 cytotoxicity in multiple myeloma cells and animal models discussed here show that > BT062 has highly significant anti-myeloma activity at doses that are well tolerated in a murine model.

A study of a single repeated dose, with a dose scale, open phase I label in patients with relapsed or relapsing / refractory multiple myeloma is performed.

The immunoconjugates described herein can be administered by any route, including intravenously, parenterally, orally, intramuscularly, intrathecally or as an aerosol. The mode of supply will depend on the desired effect. A person with skill will easily know the best route of administration for a particular treatment in accordance with the present invention. The appropriate dose will depend on the route of administration and the indicated treatment and can be readily determined by a person skilled in the art in view of the current treatment protocols.

The pharmaceutical compositions containing the immunoconjugate of the present invention and / or any additional cytotoxic agent as active ingredients can be prepared according to conventional pharmaceutical formulation techniques. See, for example, Remington's Pharmaceutical Sciences, 17th Ed. (1985, Mack Publishing Co., Easton, Pa.). Typically, effective amounts of active ingredients will be mixed with an acceptable pharmaceutical carrier. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, for example, intravenous, oral, parenteral, intrathecal, transdermal or aerosol.

The anticancer combinations of the present invention may preferably already be in the form of pharmaceutical compositions or in the form of kits comprising the components of the anti-cancer combination in different containers. The components of the equipment are usually administered in combination with each other, often they are co-administered either in a combined dosage form or in separate dosage forms. These equipment may also include, for example other components, a device for managing the components or combination, a device for combining the components and / or instructions on how to use and administer the components.

For oral administration, the immunoconjugate and / or cytotoxic agent can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, fusions, powders, suspensions or emulsions. To prepare the compositions in oral dosage form, any of the usual pharmaceutical media can be employed, such as for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Due to their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, the tablets may be sugar coated or enteric coated by standard techniques. The active agent must be stable for passage through the gastrointestinal tract. If necessary, suitable agents for stable passage can be employed, and can include phospholipids or lecithin derivatives described in the literature, as well as liposomes, microparticles (including microspheres and macrospheres).

For parenteral administration, the immunoconjugate and / or cytotoxic agent can be dissolved in a pharmaceutical carrier and administered either as a solution or a suspension. Illustrative of suitable carriers are water, saline, phosphate buffered solution (PBS = Phosphate Buffer Solution), dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetable or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the targeted agent in a conjugate and / or immunoconjugate and / or cytotoxic agent is administered intracerebroventricularly or intrathecally, it can also be dissolved in cerebrospinal fluid.

Doses administered to a subject can be specified as quantity, per surface area of the subject (including humans as well as non-human animals). The dose may be administered to said subject in preferred amounts, but not exclusively from approximately 5 mg / m2 to approximately 300 mg / m2, including approximately 10 mg / m2, approximately 20 mg / m2, approximately 40 mg / m2, about 50 mg / m2, about 60 mg / m2, about 80 mg / m2, about 100 mg / m2, about 120 mg / m2, about 140 mg / m2, about 150 mg / m2, about 160 mg / m2 and about 200 mg / m2. The immunoconjugates are administered conveniently at a time or over a series of treatments. In a multi-dose regimen, these amounts can be administered once a day, once a week or once every two weeks. Dosage loading with a single high dose or in alternate form, lower doses that are administered briefly one after the other, followed by doses synchronized at longer intervals, constitute a preferred embodiment of the present invention. In a preferred embodiment, the dose timing is adjusted for a subject such that sufficient time has elapsed before a second and / or any subsequent treatment so that the previous dose is metabolized substantially, but the amount of immunoconjugate present in the subject's system still inhibits, delays and / or prevents the growth of a tumor. An exemplary "repeated single dose" regimen comprises administering immunoconjugate doses of about 10, 20, 40, 60, 80, 100, 120, 140, 160, 180 or 200 mg / m2 once every three weeks. Alternatively, a high initial dose of for example 160 mg / m2 can be followed by a maintenance dose of one, - no¬ two or every three weeks, for example about 20 mg / m2. Other combinations can be easily evaluated by the person skilled in the art. However, other dose regimens may be useful. The progress of this therapy is easily monitored by known techniques and assays. The doses may vary, among others, depending on whether they are administered for preventive or therapeutic purposes, the course of any prior therapy, the patient's medical history, the patient's disease status, the patient's tumor burden, the genetic predisposition of the patient, patient, the concomitant diseases of the patient, the stage of illness before first treatment and response to the agent directed / immunoconjugate, the side effects experienced by the patient and the discretion of the doctor in charge.

The present invention, in one embodiment, is directed to a low dose delivery regimen with rapid elimination of plasma, and in another embodiment, to a low dose delivery regimen without rapid plasma clearance for administration of immunoconjugates as set forth herein . The first regimen generally provides less than 160 mg / m2, preferably no more than about 120 mg / m2, no more than about 100 mg / m2, no more than about 80 mg / m2, including no more than about 40 mg / m2. m2, more preferably no more than about 20 mg / m2, even more preferably not more than about 10 mg / m2 in a given interval of three weeks (cycle). The range 10 mg / m2 to 120 mg / m2 results in an average daily dose of about 475 μg / m2 to about 5.71 mg / m2, or an average weekly dose of about 3.33 mg / m2 to about 40 mg / m2. In this way, average daily doses of about 400 ug / 2 to about 5.71 mg / m2, including about 500 g / m2, about 1 mg / m2, about 2 mg / m2 and about 3 mg / m2, 4 mg / m2 , 5 mg / m2 are part of the present invention and thus are average weekly doses of approximately 3 mg / m2 to approximately 40 mg / m2, including approximately 5 mg / m2, approximately 10 mg / m2, approximately 15 mg / m2, approximately 20 mg / m2, approximately 25 mg / m2, 30 mg / m2 or 35 mg / m2. These low dose administration schedules are associated with rapid elimination of plasma in the early elimination phase, ie, any time during administration until two hours after administration is complete. What distinguishes the low-dose administration regimen from other low-dose regimens is the rapid elimination of plasma, which is defined by a cmax measured during this period that is preferably less than 55%, less than 50%, less than 40%. %, or less than 30% of the theoretical cmax.

Low dose administration regimens, are at higher levels, accompanied by less rapid plasma elimination, this is plasma eliminations that exceed 55%, often 60%, 70% 80% or 90% of the theoretical cmax value, which are referred here as moderate (equal to or> 55%, but <80% of the theoretical cmax value) or slow plasma removal (equal to or> 80% of the theoretical cmax value). To these deletions, it was surprisingly found that despite the high relative concentration of the immunoconjugate in plasma, these administration regimens are still associated with tolerable toxicities. This is despite the fact that the 'level of CD138 expression in non-target cells expressing CD138, for example cells of vital organs, such as the epithelium that are not the target of any treatment, also have relatively high CD138 (analysis of immunohistochemistry with the BB4 CD138 antibody that showed that the reactivity to this antibody to the epithelium corresponds to the plasma cells of the MM patient (US Patent Publication 20070183971)). Levels of expression of CD138 in target and non-target target cells that produce equal scores (for example plus three as in the previous example) in immunohistochemical analysis are referred to herein as comparable expression levels and are part of the present invention. In an alternate embodiment, expression levels in target cells were currently consistent below the epithelium (eg, plus one or more two or more three for the epithelium). Some tumor target cells show mixed expression levels, such as some cells have an expression level of plus two and some an expression level of plus three. The average of a representative number of cells (such as 100 randomly sampled cells) will determine whether these target or target tumor cells in question fall under the definition of having levels of expression comparable or lower than that of the epithelium. These treatment regimens are generally about 120 mg / m2, but less than 200 mg / m2 over a given three-week (cycle) interval, which results in a daily dose of about 5.71 mg / m2 to about 9.52 mg / m2 or an average weekly dose of about 40 mg / m2 to about 66.67 mg / m2.

With respect to Patient 001-006, it was remarkable that this patient had progression of the disease only after finishing the treatment, reflecting the efficacy of BT062 administration (Figure 25).

A single repeated dose refers to a sequence of administrations, wherein administration following administration is considered independent of this preceding administration. Thus, in the present context, the level of immunoconjugate in the blood of a subject can be considered as equal after each administration. Each time the immunoconjugate is administered, it is expected that equal levels of immunoconjugate are initially present in the blood.

Intervals of administration between the "single doses" of the repeated single doses are defined according to the theoretical calculated average life of an isotype of an immunoconjugate in the case of BT062, IgG4.

In general, the half-life of therapeutic antibodies depends primarily on the characteristics of the antibody / its structural aspects (for example binding to Fe receptors) and the target. For example, the binding affinity of the Fe part to the neonatal receptor FcRn is affecting the half-life. By binding to FcRn in endosomes, the antibody is rescued from lysosomal degradation and recycled to the circulation, which prolongs the half-life. For an IgG4, a half-life of 15.6 (+/- 4.5) days (Alyanakian et al., 2003; Salfeld et al., 2007) has been reported. In the study referred to herein, a "repeated single dose" has been selected that has administration intervals of three weeks. However, approximately three weeks, approximately four weeks, but also approximately five or approximately six weeks are alternate intervals for repeated single doses. A reference to "approximately" refers in the context of three weeks to +/- 96 hours and in the context of four to six weeks to +/- 120 hours.

The progress of therapy is easily monitored by known techniques and trials. The dose may vary among others, depending on whether it is administered for preventive or therapeutic purposes, the course of any prior therapy, the patient's medical history, the patient's disease status, the patient's tumor burden, the patient's genetic predisposition , the concomitant diseases of the patient, the stage of illness before, first treatment and response to the directed agent / immunoconjugate, the side effects experienced by the patient and the discretion of the attending physician.

The advantages of a low dose regimen are wide range. However, the probably most significant advantage is to reduce the risk of adverse side effects. While immunoconjugates in general allow for sensitive discrimination between target and normal cells, resulting in fewer toxic side effects than most conventional chemotherapeutic drugs, many immunoconjugates are not yet completely free of side effects. Despite a superior direction, the antigen of interest in general is also expressed in non-cancerous cells whose destruction during therapy can lead to adverse side effects. In the case of CD138, the antigen in particular is expressed in epithelial cells. Also, the immunoconjugate may be subjected to processing within the body that is not related to progression in or in a target cell and a certain percentage of effector molecule may be shed in remote sites of the target cells leading to toxic side effects.

Surprisingly, it was shown that the immunoconjugate of the present invention was effective at low doses, while exhibiting clinically acceptable toxicities. Low doses in the present invention refer to doses of up to 200 mg / m2. At doses of up to at least 120 mg / m2 but in any case at doses less than 160 mg / m2, the tested immunoconjugate of the present invention also showed rapid plasma clearance in human subjects.

Tables 7 and 8 show the observed deletion.

Plasma level of BT062 (g / ml) human cmax cmax effective cmax effective Theoretical dose (cycle 1) (cycle 4) BT062 average (more average (more (mg / m2) low, higher) low; high) 10 7 1.11 n. to. 20 14 2.9 7.06 (1.66, 4.44) (6.79, 7.34) 40 27 4.31 2.51 (0.97; 9.86) (1.02; 3.68) 80 54 18.8 14.2 (13.4; 23.6) (7.4; 21) 120 81 21.4 n. to . (15.1; 28.7) 160 109 81.2 77.4 (73.7; 85.5) 200 136 82.0 n. to. (68.0; 102.4) n.a. = data not available | Table 7: Plasma concentrations after the end of infusion and average effective cmax values of plasma BT062 obtained from patients who received a single dose / repeated single dose of BT062 (first and fourth cycle). A repeated dose administration in 21-day cycles. Cmax values were obtained between 0 and 2 hours after infusion. Administration cycles: cycle 1: day 1, cycle 2: day 22; cycle 3: day 43; cycle 4: day 64 etc.

Plasma level of BT062 (ig / val) human Dosage cmax cmax Percent of theoretical BT062 effective cmax (mg / m2) (cycle 1) (n) 10 7 1.1 15% (3) 20 14 2.9 20% (4) 40 27 4.31 16% (3) 80 54 18.8 34% (3) 120 81 21.4 26.5% (3) 160 109 81.2 74.5% (4) 200 136 82.0 60% (3) Cont.

Plasma level of human BT062 (pg / ml) Dosage BT062 cmax Percent of (mg / m2) effective theoretical cmax (n) (cycle 4) 10 n. to . n. to .

I 20 7.06 49% (2) 40 2.51 9% (3) 80 14.2 26% (2) 120 n. to . n. to . 160 77.4 71% (1) 200 n. to . n. to n.a. = data not available n: number of patients Table 8: Average effective cmax values of plasma BT062 that are obtained in patients who have received a single dose / repeated single dose of BT062 (first and fourth cycles). Administration of repeated dose in cycles of 21 days. Maximum values were obtained within the first 2 hours after injection. Cmax values were obtained between 0 and 2 hours after infusion. Effective Cmax is indicated as a percentage of theoretical calculated cmax. Administration cycles: cycle 1: day 1, cycle 2: day 22; cycle 3: day 43; cycle 4: day 64 etc.

The theoretical Cmax is calculated according to the following estimated parameters: Surface area of the body of patients 1.9 m2 Weight of Patients 70 Kg Patients 40 ml / kg (Dosage administered x surface area) / body weight) Plasma Volume Although the half-life of BT062 in plasma of the treated human subjects proved to be significantly lower than the plasma half-life observed in macaque monkeys (days) and in human plasma ex vivo (14 days), the immunoconjugate still showed efficacy in human subjects, even at administrations as low as 20 mg / m2. This fact suggests an accelerated tumor direction and binding of tumor cells, resulting in increased efficacy. This property of the immunoconjugates of the present invention probably results from the IgG4 isotype of the targeted molecule / antibody.

As noted above, an unusual rapid elimination of plasma from MM patients treated is ) observed in the early elimination phase (during infusion and approximately 0 to 2 hours after infusion) followed by a generally normal terminal elimination phase at dose levels of up to 120 mg / m2, while a more typical elimination profile was observed for all 4 patients at doses of 160 mg / m2 and 200 mg / m2 (3 patients), even though the elimination was still below the theoretical cmax value. In addition, in the administration regimens that showed rapid elimination of plasma in the early elimination phase, (for example 20, 40, 80 and 120 mg / m2) not only was rapid elimination of plasma observed in the early elimination phase but it was also He observed one. response (decreased protein M urine) including responses that are manifested in a decrease in protein M urine by more than 50% after single repeated doses (Figure 24).

Figure 17 illustrates the rapid removal of plasma for doses in the range of 40 mg / m2 to 120 mg / m2, while higher doses as illustrated herein by a dose of 160 mg / m2, showed plasma elimination closer to the value theoretical. See Figure 27 for removal of plasma that is observed at a dose of 20 mg / m2. Figure 18 compares the plasma profile of BT062 to monkeys treated at comparable doses. The comparison clarifies that the rapid elimination of plasma at low doses can not be deduced from available animal models and appears to be specific for humans. Figure 22 clarifies that the rapid elimination of plasma can not be attributed to a buffering effect caused by soluble CD138. Figure 19 shows the measured cmax values of BT062 compared to the theoretical cmax values.

At higher doses, for example 160 mg / m2, which however are relatively low doses compared to the administration schemes of other immunoconjugates, the terminal deletion profiles were closer to normal, this is closer to the theoretical cmax values . However, a rapid reduction of CRF in the serum can be observed after only a single administration, which manifests itself in a partial response after the second, third and fourth administrations (Figure 26).

Thus, in one embodiment, the invention is directed to a low dose administration regimen, such as a repeated single dose regimen, wherein a response of preference is observed at least one MR preferably VGPR, CR, sCR or PR.

The invention is also directed to a low dose treatment regimen, such as a repeated single dose administration regimen, wherein stable disease is achieved over multiple treatment cycles lasting more than 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, 30, 40, 50, 60, 70, 80, 90, 100 or more weeks.

Analogs and Derivatives A person skilled in the art of therapeutic agents, such as cytotoxic agents, will readily understand that each of these agents described herein can be modified such that the resulting compound still retains the specificity and / or activity of the starting compound. The person skilled in the art will also understand that many of these compounds can be used in place of the therapeutic agents described herein. In this manner, the therapeutic agents of the present invention include analogs and derivatives of the compounds described herein.

For illustrative purposes of the uses of the immunoconjugates, some non-limiting applications will now be seen and illustrated.

Materials and methods Construction of Chimeric Antibody (cB-B4: nBT062) B-B4 Murine antibody B-B4 as previously characterized (Wijdenes et al., Br J Haematol., 94 (1996), 318) was used in these experiments.

Cloning and expression of B-B4 and cB-B4 / nBT062 Standard recombinant DNA techniques were performed as described in detail in textbooks as for example in J. Sambrook; Molecular Cloning, A Laboratory Manual; 2nd Ed. (1989), Cold Spring Harbor Laboratory Press, USA, or as recommended by manufacturers' instructions in the case where equipment is used. PCR cloning and modification of mouse variable regions were performed using standard PCR methodology. Primers indicated in the respective results section have been employed.

Expression of cB-B4 / nBT062 COS cells of exponential growth, grown in DMEM supplemented with 10% FCS, 580 μ? / ??? of L-glutamine, 50 Units / ml penicillin and 50 g / ml streptomycin were harvested by trypsinization and centrifugation and washed in PBS. The cells were resuspended in PBS at a final concentration of lx107 cells / ml. 700 μ? of cell suspension COS was transferred to a Gene Pulser cell and mixed with heavy and kappa light chain expression vector DNA (10 μg each or 13 μg of Supervector). Cells were electroporated at 1900 'V, 25] iF using a Bio-Rad Gene Pulser. Cells transformed were cultured in DMEM supplemented with free 10% gamma globulin FBS, 580 g / ml L-glutamine, 50 Units / ml penicillin and 50 μg / ml streptomycin for 72 hours before cell culture supernatants were harvested which contains antibody.

Capture ELISA to measure expression levels of cB-B4 / nBT062 96-well plates were coated with aliquots of 100 μ? 0.4 μg / ml · goat antihuman IgG antibody diluted in PBS (4 ° C, overnight). Plates were collected three times with 200 μ? / ???? of shock absorber t (PBS + 0.1% Tween-20). The wells were blocked with 0.2% BSA, 0.02% Tween-20 in PBS, before addition of 200 μ? of cell culture supernatants containing the secreted antibody (incubation at 37 ° C for one hour). The wells were washed six times with wash buffer, before detection of antibody bound with goat antihuman kappa light chain peroxidase conjugate.

Purification of cB-B4 / nBT062 from cell culture supernatants The cB-B4 antibody was purified from supernatants of transformed COS 7 cells using the Protein A ImmunoPure Plus kit (Pierce, Rockford, IL), according to the manufacturer's recommendation.

Linkage and competition assay of cB-B4 Linkage activity analysis of B-B4 and cB-B4 a CD138 was performed using Diaclone (Besangon, France) sCD138 equipment according to the manufacturer's recommendations, considering the changes described in the results section.

RNA preparation and cDNA synthesis Hybridoma B-B4 cells were developed and processed using the Qiagen Midi kit (Hilden, Germany) to isolate RNA following the manufacturers protocol. Approximately 5 g of B-B4 RNA undergo reverse transcription to produce B-B4 cDNA using the 1st strand synthesis kit from Amersham Biosciences (Piscataway, NJ) following the manufacturer's protocol.

Cloning of immunoglobulin cDNA B-B4 Immunoglobulin heavy chain cDNA (IgH) is amplified by PCR using the MHH primer IgH (5 '-ATGG-GCATCAAGATGGAGTCACAGACCCAGG-3') [SEQ ID NO: 3] 0.02% and the region primer IgGl constant MHCG1 (51 -CAGTGGATA-GACAGATGGGGG-3 ') [SEQ ID NO: 4]. Similarly, immunoglobulin light chain (IgL) was amplified using the three different Ig primers MKV2 (51 -ATGGAGACAGACACACTCCTGC-TATGGGTG-3 ') [SEQ ID NO: 5], MKV4 (5' -ATGAGGGCCCCTGCTCA-GTTTTTTGGCTTCTTG-3 ') [SEQ ID NO: 6] and MKV9 (5 '-ATGGTATCCAC-ACCTCAGTTCCTTG-3') [SEQ ID NO: 7], each in combination with MKC primer (5 '-ACTGGATGGTGGGAAGATGG-3') [SEQ ID NO: 8]. All the amplification products were ligated directly with the pCR2.1-TOPO vector using the TOPO-TA equipment (Invitrogen, sbad, CA) according to the manufacturer's instructions.

E. coli TOP10 bacteria (Invitrogen) transformed with ligated pCR2.1 vector constructs were selected on LB-ampicillin-Xgal agar plates. Small-scale cultures were inoculated with simple white colonies, developed overnight and plasmids were isolated using the QIAprep Spin Miniprep kit according to the instructions of the manufacturers.

Determination of cDNA sequence Plasmids were sequenced using the BigDye Termination v3.0 Cycle Sequencing Ready Reaction Kit (ABI, Foster City, CA). Each selected plasmid was sequenced in both directions using primers 1210 and 1233 in cycle on a GeneAmp9600 PCR machine. The electrophoretic sequence analysis was performed on an ABI capillary sequencer.

The complete cycle of cloning RT-PCR and analysis of DNA sequences was repeated to obtain three completely independent sets of sequence information for each immunoglobulin chain.

.. DNA sequence B-B4 VK Synthesis of 1st strand is done in three independent reactions. PCR products generated using the KC and MKV2 primers (sequences given above) were ligated into pCR2.1-TOPO vectors according to the manufacturer's instruction. Clones from each independent set of RT-PCR reactions were sequenced in both directions. Sequence of primed product MKV2 was highly similar to sterile kappa transcripts originating from the myeloma fusion partner such as MOPC-21, SP2 and Ag8 (Carroll et al., Mol Immunol., 25 (1988), 991; Cabilly et al. ., Gene, 40 (1985); 157) and therefore discarded.

PCR products using MKC with primers MKV4 and MKV9 were similar to each other and differ only in the oscillation positions within the leader sequence primer.

Sequence B-B4 VH DNA Synthesis of 1st strand was performed in three independent reactions and PCR products were cloned and sequenced from each 1st strand product. Five clones were sequenced from each 1st strand.

Construction of chimeric cB-B4 expression vectors The construction of the chimeric expression vectors involves adding a convenient leader sequence to VH and VK, preceded by a Bamñl restriction site and a Kozak sequence. The Kozak consensus sequence is crucial for the efficient translation of a variable region sequence. It defines the correct AUG codon from which a ribosome can initiate translation, and the most critical simple base is adenine (or less preferable a guanine) at position -3, upstream of the AUG start. The leader sequence is chosen as the most similar sequence in the Kabat database (Kabat et al., NIH National Technical Information Service). These additions are encoded within the direct primers (For) (both having the sequence 5 '-AGAGAAG-CTTGCCGCCACCATGATTGCCTCTGCTCAGTTCCTTGGTCTCC-3' [SEQ ID NO: 9], restriction site is underlined, Kozak sequence with bold). In addition, the construction of the chimeric expression vectors involves introducing a 5 'fragment of the human gamma 1 constant region, to a natural Apal restriction site, contiguous with the 3' end of the B-B4 region and, for the light chain , add a combination donor site and the Hindl II site. The combination donor sequence is important for the correct framing of the variable region to its proper constant region, thus cutting the V: C intron. The kappa + CK intron is encoded in the expression construct downstream of the B-B4 VK sequence. Similarly, gamma-4 CH is encoded in the expression construct downstream of the B-B4 VH sequence.

The B-B4 VH and VK genes were first carefully analyzed to identify any undesired combination donor sites, combination acceptor sites, Kozak sequences and for the presence of any extra sub-cloning restriction sites that would subsequently interfere with sub-cloning. and / or functional whole antibody expression. An undesired HindIII site was found in the VK sequence that was necessarily removed by site-directed mutagenesis by PCR without changing the amino acid sequence. For these reactions, oligonucleotide primers BT03 (51 -CAACAGTATAGTAAGCTCCCTC-GGACGTTCGGTGG-3 ') [SEQ ID NO: 10] and BT04 (5' -CCACCGAACG-TCCGAGGGAGCTTACTATACTGTTG-3 ') [SEQ ID NO: 11] were used and mutagenesis was performed according to the protocol of the Mutagenesis Stratagene team (La Jolla, CA) Quickchange.

Kappa chain chimerization primers The unambiguous VK B-B4 leader sequence, independent of the PCR primer sequence, was aligned with the murine leader sequences in the Kabat database. The closest correspondence for leader B-B4 VH was VK-10 ARS-A (Sanz et al., PNAS, 84 (1987), 1085). It is predicted that this leader sequence will be correctly cut by the SignalP algorithm (Nielsen et al., Protein Eng, 10 (1997); 1). CBB4Kfor primers (see above) and g2258 (51-CGCGGGATCCACTCACGTTTGATTTCCAGCTTGGTGCCTCC-3 '[SEQ ID NO: 12]; Restriction site is underlined) were designed to generate a PCR product containing a complete leader, the B-B4 VK region, and terminal restriction sites HindIII and BamRI, to clone into the expression vector pKNlOO. The forward primer, CBB4K introduces a HindIII restriction site, a Kozak translation initiation site and the leader sequence VK-10 ARS-A. The reverse primer g2258 introduces a combination donor site and a BamUI restriction site. The resulting fragment was cloned into the Hindlll / Bamñl restriction site of pKNlOO.

Heavy chain chimerization primers The unambiguous leader sequence B-B4 VH, independent of the PCR primer sequence, was aligned with the murine leader sequence in the Kabat database. The closest correspondence for leader B-B4 VK was VH17-1A (Sun et al., PNAS, 84 (1987), 214). This leader sequence is predicted to be cut correctly by the SignalP algorithm. Primers cBB4Hfor (see above) and g22949 (5 '-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACTGAGGTTCC-3' [SEQ ID NO: 13]; Restriction site is underlined) were designed to 'generate a PCR product containing a leader VH17-1A, region B- B4 VH and terminal restriction sites tfindlll and Apal, to clone into the expression vector pG4D200. The direct primer cBBHFor introduces a HindIII restriction site, a Kozak translation initiation site and the leader sequence VH17-1A. The reverse primer g22949 introduces the 5 'end of the 4C gamma region and a natural Apal restriction site. The resulting fragment was cloned into the HindIII / Apal restriction site of pG4D200, resulting in vector pG4D200cBB.

Production of cBB4 antibody One ampule of COS 7 cells was thawed and developed in DMEM supplemented with 10% Fetal Clone I serum with antibiotics. One week later, cells (0.7 ml to 107 cells / ml) were electroporated with pG4D200cBB4 plus pKN100cBB4 (10 / g DNA each) or without DNA. The cells were coated in 8 ml of culture medium for 4 days. The electroporation was repeated seven times.

Detection of chimeric antibody A sandwich ELISA is used to measure antibody concentrations in COS 7 supernatants. Transiently transformed COS 7 cells were treated approximately 6956 ng / ml antibody (data not shown).

Link activity of cB-B4 To test the binding activity of cB-B4 in COS 7 culture supernatants, the Diaclone sCD138 kit has been employed, a solid-phase sandwich ELISA. A monoclonal antibody specific for SCD138 has been coated onto wells of the microtitre strips provided. During the first incubation, sCD138 and biotinylated antibody B-B4 (bio-B-B4) are simultaneously incubated in conjunction with a series of unlabelled test antibody dilutions (B-B4 or cB-B4).

The concentrations of bio-B-B4 in this assay have been reduced in order to obtain competition with low concentrations of unlabeled antibody (concentration of cB-B4 in cell culture supernatants COS 7 were otherwise too low to obtain sufficient competition ). Results of this test reveal that both antibodies have the same specificity for CD138 (data not shown).

Purification of cB-B4 B-B4 chimeric was purified from supernatants of COS 7 cells using the Protein A 10 ImmunoPure Plus kit (Pierce), according to the manufacturer's recommendation (data not shown).

Determination of! ¾: Comparison nBT062 / BB4 Purification of soluble CD138 "Soluble CD138 antigen of cell culture supernatant U-266 was purified by FPLC using 1 mL" HiTrap activated NHS HP "column coupled with B-B4 Cell culture supernatant was loaded in PBS buffer pH 7.4 on the column and later on the antigen CD138 was eluted with 50 mM triethylamine pH 11 in 2 mL fractions 20. CD138 eluted was neutralized immediately with 375 μL of 1 M Tris-HCl, pH 3 to avoid structural and / or functional damage.

Biotinylation of CD138 Sulfo-NHS-LC (Pierce) was used to label CD138. NHS activated biotins reacted efficiently with primary amino groups as lysine residues in buffers pH 7-9 to form stable amide bonds.

For biotinylation of CD138, 50 μ? of CD138 are desalted using centrifugation-protein desalification columns (Pierce). The biotinylation reagent (EZ-Link Sulfo NHS-LC-Biotin, Pierce) was dissolved in deionized H20 cooled by ice to a final concentration of 0.5 mg / mL. The biotinylation reagent and the capture reagent solution were mixed having 12 times the molar excess of biotinylation reagent compared to the capture reagent (50 pmol CD138 to 600 pmol of biotinylation reagent) and incubated 1 h at room temperature while The vial is gently shaken. The unbound biotinylation reagent was removed using protein desalting columns.

Immobilization of bCDl38 The sensorchip (SENSOR CHIP SA, BIACORE AB) used in the BIACORE assay is designed to link biotinylated molecules for interaction analysis in BIACORE systems. The surface consists of a carboxymethylated dextran matrix pre-immobilized with streptavidin and ready for high affinity capture of biotinylated ligands. The immobilisation of bCD138 was carried out in SENSOR CHIP SA using a flow rate of 10 .L / min by manual injection. The chip surface was conditioned with three consecutive injections of 1 minute of NaCl 1 in 50 mM NaOH. Then biotinylated CD138 was injected for 1 minute.

Determination of KD of different antibodies using BIACORE The BIACORE C software or program uses predefined masks, so-called "Installation Wizards" for different experiments where only certain settings can be changed. Since BIACORE C was originally developed to measure concentrations, there is no installation assistant designed to carry out affinity measurements. However, with the appropriate settings, the installation wizard for "non-specific link" can be used to measure affinity velocity constants and therefore was used for KQ determination. With this installation assistant / two flow cells were measured and the dissociation phase was adjusted to 90 s when performing "Regeneration 1" with cushion buffer solution BIACORE. "Regeneration 2" which is equivalent to the actual regeneration, was performed with 10 mM glycine-HCl pH 2.5. After this step, the CD138 ligand was in its competent binding state again. During the entire procedure, HBS-EP is used as a running buffer and dilution solution. To determine the binding of the different antibodies (~ 150 kDa) to CD138, association and dissociation are analyzed at different concentrations (100, 50, 25 12.5, 6.25 and 3.13 nM). The dissociation equilibrium constants were determined by calculating the velocity constants ka and kd. Subsequently, the KD values of the analytes were calculated by the coefficient of kd and ka with the BIAevaluation program. The results are shown in Table 9.

Affinity Antibody KD (nM) Average KD (nM) 1. 4 nBT062 .. 1.4 1.4 +/- 0.06 1. 5 1. 7 B-B4 1.7 1.6 +/- 0.06 1. 6 1. 9 X1BT062-SPDB-DM4 1.9 1.9 +/- 0.00 < 1.9 2. 6 B-B4-SPP-DM1 2.7 2.6 +/- 0.06 2.6 Table 9: Comparative analysis of KD values of nBT062 and? -? . Standard definitions are given for average KD values.

Discussion Average KD values for each antibody were calculated from three independent experiments. The results show that all measures nBT062 exhibits slightly diminished KD values compared to B-B4 (average KD values were 1.4 and 1.6 nM, respectively).

Preparation of Immunoconjugates nBT062-DMl and huC242-DMl The maytansinoid containing thiol DM1 is synthesized from the microbial fermentation product P-3, as previously described by Chari (Chari et al., Cancer Res. 1 (1992), 127). Preparation of humanized C242 (huC242) (Roguska et al., PNAS, 91 (1994), 969) has been previously described. Antibody-drug conjugates were prepared as previously described (Liu et al., PNAS, 93 (1996), 8618). An average of 3.5 DM1 molecules was bound per antibody molecule. nBT062-DM4 BT062 is an antibody-drug conjugate coed of the cytotoxic maytansinoid drug DM4, linked by disulfide bonds via a linker to the chimerized monoclonal antibody nBT062. Maytansinoids are anti-mitotic that inhibit tubulin polymerization and microtubule assembly or structure (Remillard et al., Science 189 (1977), 1002). Chemical and schematic representations of BT062 (nBT062-DM4) are illustrated in FIGURES 1 and 2.

Synthesis of DM4 DM4 is prepared from the well-known maitansinol derivative (Kupchan et al., J. Med. Chem., 21 (1978), 31). Maitansinol is prepared by reductive cleavage of the ester portion of the microbial fermentation product, ansamitocin P3, with trimethoxyaluminum lithium hydride (see FIGURE 3).

DM4 is synthesized by acylation of maytansinol with N-methyl-N- (4-methidithiopentanoyl) -L-alanine (side chain DM4) in the presence of dicyclohexylcarbodiimide (DCC) and zinc chloride to give the maytansinoid-containing disulfide DM4 -Sme . DM4-SMe is reduced with dithiothreitol (DTT) to give the desired thiol-containing maytansinoid DM4 (see FIGURE 4 for the process flow diagram DM4).

Immunoconjugate BT062 The procedure for the preparation of nBT062-DM4 is outlined in FIGURE 5. The nBT062 antibody is modified with N-succinimidyl-4- (2-pyridyldithio) butyrate (linker SPDB) to introduce dithiopyridyl groups. DM4 is mixed with the modified antibody at a concentration exceeding the equivalents of dithiopyridyl groups. The BT062 conjugate is formed by a disulfide exchange reaction between the thiol group of DM4 and the dithiopyridyl groups introduced into the antibody by the linker. Purification by chromatography and diafiltration removes the low molecular weight reagents (DM4) and the reaction products (thiopyridine), as well as aggregates of conjugated antibody, to produce the drug substance in bulk.

FACS analysis and WST cytotoxicity assays FACS analysis OPM-2 cells are plasma cell leukemia cell lines that show high expression CD138. OPM-2 cells were incubated with nBT062, nBT062-SPDB-DM4, nBT062-SPP-DM1"or nBT062-SMCC-DM1 at different concentrations (indicated in Figure 6) .The cells were washed and antibody bound to CD138 or conjugate was detected using a secondary antibody labeled by fluorescence in FACS analysis.The average fluorescence measured in these experiments was plotted against the antibody concentration.

Cell viability assay CD138 + MOLP-8 cells were seeded in flat bottom plates at 3000 cells / well. CD138 control cells "BJAB were seeded at 1000 cells / well.The cells were treated with nBT062-SPDB-DM4, nBT062-SPP-DM1 or nBT062-SMCC-DM1 at different concentrations (indicated in Figure 7) for five days. WST reagent (water soluble tetrazolium salt, ROCHE) is added in order to measure cell viability according to the manufacturer's instructions (ROCHE) The reagent was incubated for 7.5 hours in MOLP-8 cells and for 2 hours in BJAB cells The fraction of surviving cells is calculated based on the optical densities measured in a microplate reader using standard procedures.

Discus Link of nBT062-SPDB-DM4, nBT062-SPP-DMl, nBT062-SMCC-DMl or nBT062 is analyzed by FACS. CD138 + OPM-2 as target or target cells are incubated with nBT062 or immunoconjugated and "cell-bound" molecules are detected using a secondary antibody labeled by fluorescence. In Figure 6, the average bloom as measured for the amount of antibody bound to cells is plotted against different concentrations of conjugate or antibody. The results show that nBT062-SPDB-DM, nBT062-SPP-DM1 and nBT062-SMCC-DM1 have very similar binding characteristics. In addition, the results strongly suggest that the binding characteristics of the unconjugated antibody are not affected by the conjugated toxins.

In cell viability assays, the cytotoxic activity of the antibody against target cells or target CD138 + MOLP-8 and control cells of B-cell lymphoblast CD138"BJAB were analyzed. Both cell lines were seeded in flat bottom plates and incubated with increasing concentrations of The immunoconjugates.Unconjugated antibody is used as a control.The cytotoxic activity is analyzed five days after the addition of the immunoconjugates when using WST reagent in order to measure cell viability.In Figure 7 (A) - (C) , the fraction of surviving cells relative to control cells treated with vehicle control is plotted against increasing concentrations of immunocontained.The results show that the cytotoxic activity of nBT062-SPDB-DM4, nBT062-SPP-DMl and nBT062-SMCC-DMl against MOLP-8 cells is very similar.As expected, CD138 control cells "BJAB were not killed by the immunoconjugates, indicating that all immunoconju- nugados act by specific binding of cells to CD138. In competition experiments, where MOLP-8 cells were preincubated with a molar excess of unconjugated nBT062. Preincubation substantially blocked the cytotoxicity of nBT062-SPDB-DM4, providing further evidence that immunoconjugates kill the cells by specific binding to CD138 on the cell surface (Figure 7 (D)).

Mouse xenograft experiments To evaluate the importance of target-inducing CD138 in anti-tumor activity of antibody-maytansinoid conjugates of a human chimeric ver of antibody B-B4, nBT062, mouse xenograft experiments were performed. Two vers of maytansinoid-nBT062 conjugates were prepared that may differ in the chemical stability of their disulfide bonds (nBT062-SPP-DM1 and nBT062-SPDB-DM4). The antitumor activity of these antibody-drug conjugates was compared with the activity of the B-B4-SPP-DM1 conjugate (comprising the murine precursor antibody), as well as free conjugated maytansinoid (DM4), native unmodified nBT062 antibody, and conjugate maytansinoid-IgGl (irrelevant) without a target. The conjugates were evaluated in a positive CD138 xenograft model (MOLP-8) of human multiple myeloma in severely combined immunodeficiency mice (SCID = Severe Combined Immunodeficient).

In these mice, subcutaneous tumors (CB.17 female SCID mice) were established by inoculation with MOLP-8 cell suspens. Treatment with intravenous injection of a single bolus was performed when tumor volumes reached an average of 113 mm3. Changes in tumor volume and body weight were monitored twice a week. The experiments were carried out about 68 days after inoculation of the tumor cells.

Mouse xenograft experiments A Mice Female mice CB.17 SCID, five weeks old were obtained from Charles River Laboratories.

Human tumor cell lines MOLP-8, a human multiple myeloma cell line, is supplied by ATCC. MOLP-8 cells expressing the CD138 antigen on their cell surface and developed xenograft tumors in SCID mice, were maintained in RPMI-1640 medium supplemented with 4 mM L-glutamine (Biowhittaker, Walkersville, D), 10% fetal bovine serum (Hyclone, Logan, Utah) and 1% streptomycin / penicillin at 37 ° C in a humidified atmosphere containing 5% C02.

PART I Tumor growth in mice Each mouse was inoculated with lxlO7 MOLP-8 cells subcutaneously in the area under the right shoulder. The total volume was 0.2 ml per mouse, where the ratio of serum free medium to matrigel (BD Bioscience, Bedford, MA) was 1/1 (v / v). Before treatment, xenograft tumors were monitored daily and allowed to settle. Tumor volume reached approximately 113 mm3 approximately 11 days after inoculation of tumor cells. The tumor capture rate of CB.17 SCID mice was 100%. 11 days after inoculation of tumor cells, 42 mice were selected based on tumor volumes and body weights. The tumor volume was in the range of 68.2 to 135.9 mm3. Forty-two mice were randomly divided into seven groups (A-G) of six animals each based on tumor volume.

Each of six mice in Group A received 200 μ? of PBS as vehicle control. Each mouse in group B received 13.8 mg / kg of nBT062 of naked antibody. This dose is equivalent to the amount of antibody component nBT062 at 250 μg / kg bound maytansinoid. The ratio of molecular weights of maytansinoids to antibody nBT062 in a conjugated molecule is approximately 1/55. Each mouse in Group C received 250 μg / kg of DM4. Each mouse in Group D received 250 g / kg of huC242-DM4. Mice in groups E, F and G received 250 Ug / kg of nBT062-SPDB-DM4, B-B4-SPP-DM1 and nBT062-SPP-DM1 each respectively.

All agents were administered intravenously as a single bolus injection through a lateral vein of the tail with a 1 ml syringe adapted with a 27 gauge 1.27 cm (½ inch) needle. Before administration, stock solutions of antibody nBT062, nBT062-SPDB-DM4 and nBT062-SPP-DM1 were diluted with sterile PBS at concentrations of 2 mg / ml, 28.1 μg ml and 28.1 pg / ml, respectively, in such a way that the volume injected by each mouse was between 120-220 μ? .

PART IX In a second set of experiments, cells MOLP-8 (1.5xl07 cells per mouse), suspended in a 50:50 mixture of serum free medium and matrigel were injected subcutaneously in the area under the right shoulder in 100 μ ?. Tumor volumes reached approximately 80 mm3 on day 11 and the average of the controls was approximately 750 mm3 on day 25, after cell inoculation. The times of tumor duplication were estimated as 4.58 days. Each mouse in the control group (n = 6) received 0.2 ml of sterile PBS administered in the lateral tail vein (i.v.) in a bolus injection. All treatment doses were based on conjugated maytansinoid. Nine groups (n = 6) were treated with a single intravenous injection of nBT062-SMCC-DM1, nBT062-SPDB-DM4, or nBT062-SPP-DM1, each at doses of 450, 250 and 100 μg / kg. An additional group (n = 6) received 250 ug / kg of nBT062-SMCC-DM1 in a repeated dose (weekly for five weeks). Mice were randomized into 11 groups (n = 6) by tumor volume using the LabCat Program. Tumor volumes were in the range of 40.0 to 152.5 mm3. The mice were dosed based on individual body weight.

Tumor size was measured twice a week in three dimensions using the LabCat System (Tumor Measurement and Tracking, Innovative Programming Associated, Inc., Princeton, NJ). The volume of tumor in mm3 was calculated using the methodology described in Tomayko et al. (Tomayko et al., 1989): Volume = Length x Width x Height x ½ Logium cell extermination was calculated with the formula described by Bissery et al. (Bissery et al., 1991). cellular extermination Logio = (T-C) / Td x 3.32 Y wherein (T-C) or retardation in tumor growth, is the average time in days required for the tumors of the treatment group (T) and the control group (C) to reach a predetermined size (600 mm3). Td is the tumor doubling time, based on the average tumor volume in the control and 3 mice. 32 is the number of cell duplications per log of cell growth.

Results Tumor growth in individual mice is shown in Figures 8 and 9. The average tumor growth (+/- SD) for each group is shown in Figure 10.

Compared to tumor growth in the PBS-treated animals, treatment with nBT062 antibody, unconjugated free DM4 or the irrelevant non-targeting conjugate huC242-D4 does not cause any significant inhibition of tumor growth.

All three conjugates directed to CD138, nBT062-SPDB-DM4, B-B4-SPP-DM1 and · nBT062-SPP-D at a dose of 250 μl / 1 ^ caused a marked delay in tumor growth. Based on the average tumor volumes measured in the treatment groups, the DM4 conjugate nBT062-SPDB-DM4 was the most active, while the nBT062-SPP-DMl conjugate showed slightly increased activity compared to its murine counterpart B-B4 -SPP-DM1 (Figure 10). The results obtained in individual mice also show that the antitumor activity obtained with B-B4-SPP-DM1 is more heterogeneous and less predictable than that measured in mice treated with nBT062-SPP-D 1. In terms of homogeneity of antitumor activity, the another conjugate that uses nBT062 as directed antibody nBT062-SPDB-D 4 behaved similar to nBT062-SPP-DMl.

No reduction in body weight was observed in any treatment group suggesting that the treatments were well tolerated.

Discussion The results of the analysis of three conjugates directed to CD138 in experimental animals demonstrated the importance of targeted delivery for antitumor activity. While the maytansinoid conjugates of human chimeric nBT062 and murine B-B4 antibodies show significant activity as measured by log cell extermination, there was no significant impact on tumor growth from treatment with unconjugated DM4, unmodified native huBT062 antibody , or unguided control conjugate (huC242-DM).

The immunoconjugate prepared from the human chimeric antibody, nBT062-SPP-DM1 gave a slightly higher antitumor activity than the conjugate prepared from its murine counterpart B-B4-SPP-DM1. In addition, treatment with nBT062-SPP-DM1 and nBT062-SPDB-DM4 resulted in more homogeneous responses in individual mice compared to treatment with B-B4-SPP-DM1. The high binding variation of B-B4-SPP-DM1 explained that measurement of average tumor volume (+/- SD) of MOLP-8 human multiple myeloma xenografts in CB.17 SCID mice with time (days) later inoculation currently gave relatively better results for B-B4-SPP-DM1 than for nBT062-SPP-DM1 (data not shown). This characteristic of immunoconjugates using nBT062 as a directed antibody appears to be beneficial especially for therapeutic use of conjugates.

Finally, the most potent of the maytansinoid conjugates after simple IV administration in the MOLP-8 xenograft models in SCID mice was nBT062-SPDB-DM4.

Extermination due to non-specific activation (cell viability test) CD138 + OP 2 cells and CD138"Namalwa cells were seeded in round bottom plates either in separate wells or in co-culture.The cells were treated with nBTO 62-SPDB-D 4 in concentrations in the range of lxlO" 8 to lxlO -9 M. The fraction of viable cells was detected using the WST reagent (water soluble tetrazolium salt, ROCHE) according to the manufacturer's instructions (ROCHE). The fraction of surviving cells is calculated based on the optical densities measured in a microplate reader using standard procedures.

Discussion Extermination by nonspecific activation of non-targeted cells in immediate proximity (as in round-bottomed wells) to multiple myeloma cells under treatment with nBT062-SPDB-DM4 was analyzed in an in vitro study in which 0PM2 CD138 positive cells were cultured in co-culture with negative CD138 Namawla cells (Figure 13). In general, whereas CD138 positive cells are efficiently killed by nBT062-SPDB-DM4, negative CD138 cells were not affected by the conjugate. In co-culture in round bottom wells, however nBT062-SPDB-DM4 also killed antigen-negative cells in immediate proximity to positive antigen cells (an effect often referred to as non-specific activation killing). Kovtun et al. (2006) argued that extermination by nonspecific activation mediated by maytansinoid conjugates occurs only in immediate proximity to positive antigen cells. Kovtun et al. (2006) which is incorporated herein by reference in its entirety, also discusses the importance of the immunocolator linkage. Extermination by nonspecific activation in vivo may contribute to) the eradication of tumor cells that express in heterogeneous form CD138, 2) the destruction of the tumor microenvironment by the extermination of tumor stromal cells, and 3) the prevention of the selection of resistant cells nBT062-SPDB-DM4 CD138 negative.

The effect by nonspecific activation is of particular importance if the activity of an immunoconjugate is impaired by the targeted antigen that is expressed in tumors in a heterogeneous form. If this is the case, a particular cell of a tumor expresses if it does in fact, the antigen not in a quantity that would allow targeted or direct direct targeting and extermination of the cell by the respective immunoconjugate. The antitumor efficacy of nBT062-SPDB-DM4 in CD138 negative cells in co-culture with CD138 positive cells clarifies that the presence of target cells influences, under the appropriate circumstances, the cytotoxic activity of nBT062-SPDB-DM4 towards targeted cells.

Mouse-xenograft B experiments In this set of experiments, eighty-five mice were inoculated with MOLP-8 cells (1.5xl07 cells / mouse) subcutaneously in the right shoulder. The rate of tumor intake was 100%. Sixty-six SCID mice containing bulky MOLP-8 tumors with an average tumor volume of approximately 80 mm3 were randomized into eleven treatment groups (n = 6). Mice were treated with a single dose of one of three conjugates (nBT062-S CC-DMl, nBT062-SPDB-DM4 or nBT062-SPP-DM1). One additional group received five weekly doses of nBT062-SMCC-DMl and one control group received a single dose of PBS. Average tumor volumes are illustrated in Figure 11A. A dose response was established for each conjugate. An average tumor volume of 750 mm3 in the animals treated with PBS was reached on day 25. Tumor doubling time determined by curve fitting with linear radiation of best fit in a log-linear trace of tumor growth control was 4.58 days. Animals treated with nBT062-SPDB-DM4 at 450 pg / kg had the highest cell extermination log (LCK = 2.89), followed by animals treated with nBT062-SMCC-DMl at 250 ug / kg weekly dose (LCK = 2.1; see Table 10). Comparison of the average tumor growth curves for the treatment groups by repeated measures ANOVA performing Dunnett's Multiple Comparison Test showed a significant difference between the control group PBS 450 μg / .g of nBT062-SPDB-DM4 (p < 0.01), 250 ug / kg of nBT062-SPDB-DM4 (p <0.05) and five-week dose "of 250 g / kg of nBT062-SMCC-DMl (p <0.05) .There was no partial tumor regression or complete in any of the treatment groups that occurred except for an animal receiving 450 g / kg of nBT062-SPDB-DM4, which had partial regression of the tumor until day 85 after inoculation.

Test material Dosage LCK Dosage (Ug / kg) PBS dose simple nBT062-SMCC-DMl 450 0.85 dose simple nBT062-SMCC-DMl 250 0.53 doses simple nBT062-SMCC-D l 100 0 dose Simple nBT062-SPDB-DM4 450 2.89 doses simple nBT062-SPDB-DM4 250 1.05 dose simple nBT062-SPDB-D 4 100 0.39 doses simple nBT062-SPP-DMl 450 0.8 dose simple nBT062-SPP-DMl 250 0.39 dose simple nBT062-SPP-DMl 100 0.2 dose Simple nBT062-SMCC-DMl 250 2.1 weekly for 5 weeks Table 10. Log extermination values Log (LCK = Log Cell Kill) as measured for antitumor activity of different nBT062-D x conjugates in different dosage schemes. Refer to the materials and methods section for information regarding calculation of LCK values.

Live Jn efficacy of nBT062-SPDB-DM4 and nBT062-SPP-DM1 in the bone marrow environment Preparation of SCID mice that have human fetal bone implants Human fetal long bones (pieces of human fetal bones) were implanted into the upper body of CB17 SCID mice (SCID-hu) as previously described (Urashima et al., 1997) and thus a mouse model is provided for the migration of human MM cells to human BM cells.

Treatment regimen (SCID-hu / INA-6 mice) 4 weeks after bone implant, 2.5xl06 INA-6 cells in a final volume of 100 // L of RPMI-1640 cell culture medium were injected directly into the cavity of human bone marrow in the SCID-hu mice described above. An increase in the levels of soluble human IL-6 receptor (shuIL-6R), which is released by INA-6 cells, is used as a parameter of MM cell growth and disease burden.

The mice developed serum shuIL-6R susceptible to be measured approximately 4 weeks after INA-6 cell injection and then received 0.176 mg of conjugate or vehicle control by tail vein injection, weekly for 7 weeks. After each treatment, blood samples were collected and measured for levels of shuIL-6R by an enzyme-linked immunosorbent assay (ELISA, R & D Systems, Minneapolis, MN). The results are illustrated in Figure 12.

Discussion Interleukin 6 (IL-6) is a growth and survival factor for multiple myeloma cells. INA-6 is a human myeloma cell line dependent on IL-6, which also requires bone marrow stromal cells (BMSC) to proliferate. INA-6 cell lines produce soluble IL-6 receptor (shuIL-6R). An increase in the levels of shuIL-6R can be used as a parameter of MM cell growth and disease burden.

In this way, sCID-hu / INA-6 mice provide a model for multiple myeloma cells that grow in their normal bone marrow environment. The tumor cells of this model, which interact directly with the human bone marrow, closely resemble the situation in patients, where tumor cell growth is also promoted by the presence of stromal cells. As INA-6 cells release soluble human interleukin-6 receptor (shuIL-6R), serum concentrations of this protein can be used as a measure for loading tumor cells in these mice. The in vivo potency of nBT062-SPDB-DM4 and nBT062-SPP-DM1 was tested in this environment.

Treatment of SCIDhu / INA-6 mice with i.v. administrations Weekly doses of nBT062-SPDB-DM4 or nBT062-SPP-DMl for seven weeks, induces efficient tumor regression, as detected by a decrease in serum shuIL-6R levels relative to the control, indicating good efficacy of the conjugates even in the environment of human bone marrow, which reflects the relevant situation in patients (Figure 12). · - Dose in Mice In order to determine relevant doses, a single administration of. BT062 at doses of 100, 250 and 450 μ? / Kq (based on the DM4 concentration) was given to the mice when the tumors reached an average tumor volume of 80 mm3 (Figure 14). Doses are reported as a DM4 concentration of conjugate (1 / ig of DM4 equal to about 55 μ of antibody protein). The anti-tumor efficacy was dose-dependent at the highest dose tested (450 / g / kg) resulting in a Log cell (LCK) kill of 2.9 (Also compare Table 10). The animals regained weight throughout the course of the study indicating that the treatment was not toxic to the mice.

Figure 14 indicates 250 / g / kg as a first - - effective dose in mice, which results in a comparable dose in humans of 166 mg / m2.

Indicator: Xenograft Models - Pancreas / Mammary and other Carcinomas General Experimental Provision In accordance with CD138 expression analysis (immunohistochemical analysis in micro rows of tumor tissue), tumor candidates were selected from a collection of primary tumors, ie, tumors derived from patients. After subcutaneous transplantation and tumor establishment (induction time 30 days), the BT062 immunoconjugate was intravenously injected at 2 different concentrations of the maytansinoid DM4, 450 μq / kg and 250 μg / kg (each based on molecular weight of bound DM4 (1 mg of DM4 is conjugated to 52 mg of antibody, equaling a total mass of 53 mg, 450 μg / kg DM4 = 23,850 // g) The immunoconjugate was administered once a week for 10 weeks ( in case of treatment in mice implanted with pancreatic tumor) and 5 weeks (in case of mice implanted with mammary tumor), Example 1: Pancreas carcinoma Pancreatic tumor tissue (PAXF 736 (Kuesters et al., 2006) was implanted (bilateral) in NMRI mice.The implanted tumor originated from a primary pancreatic carcinoma to a patient (poorly differentiated), infiltrating adenocarcinoma (an exocrine carcinoma),). No side effects were observed. The tumor of this patient was identified as tissue of high expression of CD138 by Immunohistochemistry studies. However, CD138 is not expressed to a comparable degree with myelomatous plasma cells in patients of my multiple lobe, as detected in tumorigenic cell lines by flow cytometric surface staining.

Treatment with BT062 was initiated after the tumors reached- 'size' of approximately 6-8 mm diameter (minimum 5 mm). Tumor diameters have been measured twice a week. Tumor volumes were calculated according to the formula a * b * b / 2 where "a" is the longest axis ·. and "b" its. perpendicular axis. The inhibition of tumor volumes in the test groups relative to the vehicle control group is calculated as the ratio of the median relative tumor volumes (T / C).

Inhibition of tumor for a particular day (T / C in%) is calculated from the ratio of the average RTV values (relative tumor volumes) of the test groups against control multiplied by 100%.

Average tumor volumes relative to control group Day T / C (Day x) = x 100% Average relative tumor volumes of the test group Day The tumor volume can be significantly reduced by this weekly administration of BT062. As can be seen in Figure 28, complete and partial dose-dependent remission was observed. The Figure shows that at a dose of 23.85 mg / kg, complete remission can be obtained 28 days after tumor implantation, while at a dose of 13.25 mg / kg, complete remission can be obtained 35 days after tumor implantation. Notably, after 52 days all mice in both administration regimens were still "alive" (8/8), that the eighth mouse of control group hd has been reduced to 1. A T / C value of less than 10 % indicates complete remission (CR = Complete Remission) (Bissery et al., 1991) .According to these criteria, CR is achieved in both treatment groups, reflecting the complete remission that was achieved by BT062.

Notably in a phase of observation free of treatment, no tumor regrowth is detected, confirming the complete cure in this model.

Day 52: Relative Volume of average T / C Tumor interval (%) (%) (±) Control 2055. 2055 BT062-DM4; 13. 25 mg / kg 0 (± 1.0) 0 - 3.5 0.0 BT062-DM4; 23. 85 mg / kg 0 (± 0.01) 0 - 0.1 0.0 Table 11: Volume of tumor is mouse model-pancreatic cancer xenograft Example 2: Breast carcinoma NMRI mice (nudes) were implanted (bilateral) with a patient's primary mammary tumor (determined by IHC analysis as strong positive CD138). A carcinoma-breast skin metastasis was taken in stage MI. A tumor that does not respond to Herceptin, (under Her2 with intermediate expression). The tumor was a negative estrogen receptor and a negative progesterone receptor. Tumors to be implanted were selected according to the results of IHC staining (strong, homogeneous expression of CD138 detected by BT062, triple negativity (negative expression of estrogen and progesterone hormone receptors), expression Her2 scored 2 or less (with respect to Herceptin not answer back) .

Treatment with BT062 was initiated after the tumors reached an approximate size of 6-8 mm in diameter (minimum 5 mm). Tumor data were measured twice a week. The tumor volumes were calculated according to the formula a * b * b / 2, with "a" which is the longest axis and "b" its perpendicular axis. Inhibition of tumor volumes in the test groups with respect to a vehicle control group is calculated as the ratio of the median relative tumor volumes (T / C).

The tumor volume could be significantly reduced by weekly administration of BT062. A partial and complete dose-dependent remission was observed. The immunoconjugate was well tolerated, with no influence on body weight after each injection. A lower T / C value of 10% was obtained in both treatment groups, reflecting a complete 'remission achieved' by the administration of BT062 As can be seen in Figure YYY, the anti-tumor effect (ie, complete remission) is achieved after 21 days, which can be considered a rapid response BT062.In comparison with the pancreatic model, the duration of treatment could be cut in half (5 weeks instead of 10 weeks) and the low dose of 13.25 mg / kg was reduced to 4 mg / kg to achieve a similar effect, ie complete remission and no tumor regrowth . The shorter treatment period for mammary carcinoma was not expected, since the IHC analysis level of CD138 expression was similar. In this way, no conclusions could be drawn from the level of expression CD138 to a general recommendation for the duration of treatment. After 21 days all the mice of both treated groups as well as the control group were still alive. In a period of observation free of treatment (39 days after the last administration of the immunoconjugate) no tumor regrowth was detected, confirming the complete cure.

Average tumor volume Relative T / C interval (%) (Day 21) Control (PBS) 533 (± 149.5) 339 - 878 BT062-DM4; 13. 25 mg / kg / 4 0 (± 0.02) 0 - 0.1 0.0 mg / kg BT062-DM4; 23. 85 mg / kg 0 (± 1.75) 0 - 6.6 0.0 Table 12a: Tumor volume is a mouse-xenoinj model of mammary carcinoma.

FFPE tissue samples Staining grade (membrane) 0. 25 0.05 μ g / mL μ g / mL Breast, tumor 3 Homo 2-3 Homo Mets, -061909-13 Breast, tumor 2-3 Homo 1-2 Unknown, -061909-12 Hetero Mama, tumor 3 Hetero 2 Focal Mets, -061909-09 Breast, tumor 3 Straight 1-3 Primary, -111904-4 Straight Breast, tumor 3 Straight 1 Straight Primary, -111904-1 Sample of Normal Skin 1 3 Homo 3 Homo Sample of Normal Skin 1 3 Homo 3 Homo Table 12b: Expression of CD138 in mammary carcinoma cells against epithelial cells Example 3: Bladder Carcinoma NMRI mice (nudes) are implanted with a bladder tumor (determined by IHC analysis as CD138 strong positive), ie a transition cell carcinoma.

Treatment with BT062 starts after the tumors reached a size larger than 5 mm. Tumor diameters are measured twice a week. Tumor volumes are calculated according to the formula a * b * b / 2, with "a" which is the longest axis and "b" its perpendicular axis. The inhibition of tumor volumes in a test group with respect to the vehicle control group is calculated as the ratio of the median relative tumor volumes (T / C).

Tumor volumes are significantly reduced by weekly administration of BT062. Any partial and complete dose-dependent remission is followed up.

Example 4: Pulmonary Carcinoma NMRI mice (nudes) are implanted with a lung carcinoma (determined by IHC analysis as strong positive CD138).

Treatment with BT062 starts after the tumors reached a size larger than 5 mm. Tumor diameters are measured twice a week. Tumor volumes are calculated according to the formula a * b * b / 2, with "a" which is the longest axis and "b" its perpendicular axis. The inhibition of tumor volumes in test groups relative to the vehicle control group is calculated as the ratio of mean relative tumor volumes (T / C).

Tumor volume is sought to be significantly reduced by weekly administration of BT062. Any partial and complete dose-dependent remission is followed up.

Preclinical Toxicity Studies Mouse-xenoin models are excellent for determining whether the immunoconjugates of the present invention are effective or not in the context of cancer modeled by the mouse. However, since these models lack the proper inherent expression of CD138, they can not serve as a reliable model for toxicity studies and thus can not be used to fully determine the tolerable amounts of the immunoconjugates of the present invention.

In macaque and rhesus monkeys they also have not shown that BT062 binds to any antigens related to CD138 but are currently the most suitable animal species for known BT062 toxicology studies. It is therefore expected that the toxicity profile of BT062 in mice and monkeys is due to the non-targeted effects of the cytotoxic component of the conjugate (DM4).

Toxicity studies in a single dose were carried out in compliance with GLP requirements in macaque monkeys and in CD-1 mice. These studies were designed to identify doses that cause severe toxicity and those that do not have adverse (or minimal) effects in animals, to identify potential toxicities in humans and to identify a safe initial dose for a Phase I clinical trial using an infusion of only one bolus of BT062. « Acute Toxicity Study in Mice A single-dose toxicity study was carried out in mice with BT062 administered by a single IV bolus injection in the range of 60-255 mg / m2 (20-85 mg / kg). The highest non-toxic toxic dose (HNSTD = Highest Non Severely Toxic Dose) of BT062 in mice was 45 mg / kg (135 mg / m2), with an estimated STD10 of 57 mg / kg (171 mg / m2).

Study of Acute Toxicity in Monkeys A toxicity study was performed in a single dose in macaque monkeys with BT062, administered IV at doses in the range of 48-336 mg / m2 (4-28 mg / kg). The HNSTD of BT062 in macaque monkey, was 12 mg / kg (144 mg / m2). i Human Tests with BT062 In the context of the present invention, human subjects responded well to a low dose regimen. This was even the case in the absence of any additional treatments that could compensate for potential variations in qualitative or quantitative expression of CD138 in the target cells (as compared to MYLOTARG). While mouse models demonstrated that BT062 has highly significant anti-myeloma activity at doses that are well tolerated in mice, the effectiveness was considerably better at relatively high doses (see Figure 14), raising the question of how higher doses have been tolerated by subjects humans expressing CD138 in a wide variety of non-tumor cells.

Phase I research study This study is done to test the effects (good and bad) and determine the maximum tolerated dose (MTD = Maximum Tolerated Dose) of BT062 to treat patients with multiple myeloma with relapse or with refractory relapse.

So far, 26 patients were recruited. At least 11 of 26 patients experienced decreased disease progression as represented by receiving at least one next treatment cycle, while 4 patients are still undergoing treatment. The test is performed in different sites, with groups of 3 and 4 patients treated with different dose levels (10 mg / m2, 20 mg / m2, 40 mg / m2, 80 mg / m2, 120 mg / m2, 160 mg / m2, 200 mg / m2) at any point between 1 to 10 treatment cycles (see Figure 23). As the person skilled in the art will appreciate, a greater number of treatment cycles is possible and within the scope of the present inventions, such as 10 to 50, 10 to 100, 10 to 200 and more.

Decreased progression of the disease with relatively low dose levels, ie 20 mg / m2, 40 mg / m2, 80 mg / m2 and 120 mg / m2 with a patient in the 2nd dose level at 20 mg / m2 that does not exhibits progression of the disease for 10 treatment cycles of 21 days. See Figure 23, where the patient samples 001-001, 003-001, 002-002, 002-003, 002-004, 003-003, 001-005, 001-006, 001-008, 001-009 , 004-001, 001-011, 004-002 and 001-012 it was observed that the disease progressed eventually even when it is stable disease and response, including minor and partial responses could be observed. In patient sample 001-006, the dose was maintained.

At these dose levels, as described above (see Tables 7 and 8), rapid elimination of BT062 1000 from plasma was also observed. Some pharmacokinetic profiles of these low dose administration schedules are illustrated in Figure 17. A comparison of plasma profiles of BT062 in humans with those of monkeys is shown in Figure 18. The graph on the left shows the difference at 120 mg / m2, while those on the right (160 mg / m2) show considerably smaller differences.

Doses of 160 mg / m2 and 200 mg / m2 were also administered. A dose of 16Q mg / m2 is identified as BAT and studies are expanded in this group. A dose of 200 mg / m2 is identified as MAD. Dose limiting toxicities (DLT = Dose Limiting Toxicities) were determined using a graduation in accordance with NCI CTCAE v3.0 (August 09, 2006, http://ctep.cancer.gov). Specific study DLT criteria are listed below: No Hematologic • Alopecia, of any degree is not considered DLT • Nausea grades 3-4 and vomiting that lasts more than 3 days despite optimal antiemetic medication3.

• Grades 3-4 diarrhea lasting longer than 3 days despite optimal antidiarrheal medication3. to. Optimal antidiarrheal and antiemetic treatment was determined by each investigator.

Hematological • Neutropenia grade 4 that lasts more than 5 days.

• Neutropenia grade 3 or higher with temperature greater than or equal to 38.3 ° C (101 ° F) for 2 consecutive determinations separated by 4 hours.

· Grade 4 thrombocytopenia • Thrombocytopenia grade 3 or higher with bleeding and requiring the use of platelet transfusion.

• Grade 3 neutropenia, grade 3 thrombocytopenia were NOT considered DLTs.

All adverse events (Aes = Adverse Events) were evaluated according to NCI-CTCAE v3.0. For AEs not mentioned in NCI-CTCAE v3.0, severity was estimated by the researcher according to these criteria. Only grade 1 and grade 2 were acceptable, so grade 1 (Light) requires minimal or no treatment and does not interfere with the daily activities of patients and grade 2 (Moderate) results in a low level of inconvenience or consideration with the measures therapeutic Moderate events can cause some interference with the subject's functioning.

AEs of Grade 3 (Severe) and Grade 4 (Life Threatening) considered related to BT062, were considered non-acceptable and defined as DLT, if they were not otherwise defined by DLT criteria specific to the study.

At the time samples from patients 003-005, 002-012, and 002-011 were still participants in the study.

The patients of samples 002-003, 001-002 and 002-008 were removed.

As indicated in Figure 23. Repeated simple doses of regimen 10 mg / m2, 20 mg / m2, 40 mg / m2, 80 mg / m2, 120 mg / m2, 160 mg / m2, 200 mg / m2 were performed each 21 days, which means day 1, day 22, day 43, day 64, day 85, day 106, and so on. The disease has been and will be monitored by evaluation of the doctor's hematology, clinical symptoms and clinical chemistry as well as by measuring levels of M protein in the serum and urine of patients in (g / dL) and free light chain levels (FLC = Free Light Chain) in the serum of patients over time.

Immunoglobulin Evaluation The amount of Ig antibodies including determination of IgG subgroups was analyzed in the evaluation.

Quantification of Protein M and Serum Free Light Chain Assay Initially, response to treatment was evaluated on day 1 of treatment cycles 1-3 by quantification of M protein using immunoelectrophoresis (IEP) and serum electrophoresis (IFE) immunofixation and 24-hour urine collection. For treatment cycles 3 and beyond, quantification of M protein was performed at the Day 15 visit so that the results are available to estimate response before beginning the next treatment cycle. An evaluation of general quantitative immunoglobulin was performed together with the quantification of M protein.

Serum samples were used to perform FLC assays to examine multiple myeloma subjects without detectable M protein (non-secretory / oligosecretory myeloma) and para. allow detection of early response to treatment. Therefore, serum FLC assays were performed on days 1, 2, 3, and 8 of the treatment cycle on days 1, 2, 3, 8, and 15 of cycle 4, as well as on days 1, 8, and 15 of all other treatment cycles. Protein M and FLC were analyzed in the evaluation and in the final visit. Evaluations on day 1 of cycle 1 served as reference values.

\ Dosage Figure Measurements of protein M in mg / m2 urine / serum and FLC measurements 20 Figure - Protein M in urine decreased 24 after the 3rd treatment by approximately 17 weeks and increased after the 9th treatment - M protein criterion for Minor response was reached after the 8th treatment - Decrease in Protein M level in Urine baseline by more than 50% and from Day 42 (3rd treatment) in more than 75% - Progression of diseases after Cycle 10 - Protein M in serum between 0.06 and 0.1 g / dL (defined as not measurable) 40 Figure - Stable disease for 14 weeks 25 - Protein M in serum decreased after the '1st treatment and stabilized for 14 weeks - Progression of diseases observed after treatment is maintained at. start of cycle 6 (day 105) - Protein M in urine increased from 0 in the evaluation to a maximum of approximately 16 mg / 24h (defined as not measurable) 5 160 Figure - Serum FLC level increased during 26 the evaluation period starting 21 days before day 1 of treatment - Serum FLC level decreased very 10 soon after the 1st treatment and was already close to 25% decrease on day 8.

- Compared with reference line, FLC levels are reduced 15 in approximately 40% during the 1st cycle and in more than 50% after the 2nd, 3rd and 4th treatment.

- FLC Criteria for Response Partial were reached very early. twenty - . 20 - Progression of disease after the end of the 4th cycle of treatment.

- Protein M in non-measurable serum = 0; Protein M in urine decreased from 140 mg / 24h in reference line to 25 120 mg / 24h before the 2nd treatment (defined as not measurable) = > Non-secretory myeloma Table 13 Provides observations made regarding M protein in Urine / Serum and serum FLC measurements in selected patients.

De-termination of BT062 and plasma DM4 To estimate PK properties of a single dose of BT062, after IV administration of BT062, extensive plasma sampling was performed during the first treatment cycle. The same evaluation was carried out during the treatment cycle 4. In a smaller proportion, the plasma samples were also obtained on day 1 and 8 of all the other treatment cycles, as well as at the final visit and follow-up.

Determination of Shed CD138 and HAPA All pre-dose plasma samples were evaluated for knockdown / soluble CD138 levels (sCDl38) to investigate a potential correlation between sCD138 levels and anti tumor activity. These measurements also allow determining cmax values lower than expected do not depend on the amount of sCD138 present before administration of BT062 (see Figure 22). Pre-dose plasma samples from day 1 of each treatment cycle and each final visit and follow-up, were evaluated by the presence of humoral responses against BT062 (drug product) by evaluation of anti-human product antibodies (HAPA = Human Antiproduct Antibodies ).

Measurements of CD138 detached observed In myeloma patients, high levels of SCD138 can be observed and can be an indicator of prognosis of myeloma patients (Maisnar et al., 2005).

Patients with GUS and M can exhibit high levels of soluble CD138 concomitant with higher levels of p2-rtiicroglobulin and 'high' content of plasma cells in the bone marrow (Aref et al., 2003).

A team was used to determine soluble CD138. Surprisingly, it was found that at 20 mg / m2 of BT062 in. the patient 003-003 ,. this patient exhibited a minor response regarding levels of protein M in urine, although this patient exhibited high levels of sCDl38 before treatment.

Soluble CD138 values were determined in different subjects.

Subject SCD138 (ng / mL) 002-003 61.3 001-002 196 002-004 56.7 003-003 2583 Average 724.1 - - Table 14: Patient 003-003 (dose 20 mg / m2) exhibited very high values of sCD138. However, this patient achieved a lower response at the level of Protein M.

COMBINATION STUDIES Possible antimyeloma drug candidates have been evaluated as combination partners for "BT062 in cell lines.

Cell Line Studies Combination studies in mouse-xenograft models were preceded by 'studies' in cellular 'lines'. The determination of synergy in different cell lines was made according to Chou and Tallay (1984), using the mean effect analysis. Here, IC50 values are calculated for the cytotoxic effects for each drug and each cell line, and then proportions IC50 for each pair of drugs. The cells were then exposed to dilution series of either of these drug mixtures or the drugs alone. Experimental data were analyzed using the CompuSyn program (ComboSyn, Inc., Paramus, NJ). Combination Indexes (CI = Combination Indexes) for each independent experiment were calculated and reported separately. In the analysis, CI less than 1, equal to 1 and more than 1 indicates synergy, additivity and antagonism, respectively. According to the User 's guide, 2004), the author of the method, the synergy scale and the antagonism is as follows: Description Index Combination < 0.1 Very strong synergy 0. 1-0.3 Strong synergy 0. 3-0.7 Synergy 0. 7-0.85 Moderate synergy 0. 85-0.9 Light synergy 0. 9-1.1 Almost additive 1. 1-1.2 Slight antagonism 1. 2-1.45 Moderate antagonism 1. 45-3.3 Antagonism 3. 3-10 Strong antagonism > 10 Very strong antagonism Cells RPMI 8226 MOLP8 U266 Drug Bo tezomib Slightly Antago¬ Additive nista antagonist Thalidomide Additive Additive to Antagoligeramente synergistic nista antagonist Lenalide- Antagonist Additive to Measure synergetic light to synergistic moderate Melfalan Sinergistico Antagonist Additive to additive to light to slightly moderate synergistic synergistic Dexameta- No Additive Additive is determined Table 15: Estimates of synergistic results obtained in cell lines according to the method of Chou and Talalay (1984).

In this example, lines of MOLP 8 cells were used for combination of BT062 with bortezomib, thalidomide, lenalidomide, melphalan and dexamethasone.

Combination with thalidomide or bortezomib, neither results in a synergistic or additive effect, but rather an antagonistic effect. In contrast to these cell culture studies, the combination with bortezomib was synergistic in the xenograft model described below.

Possible anti-myeloma drug candidates have been evaluated as combination partners for BT062 in Xenograft studies using human MOLP8 multiple myeloma cells.

Example 1 Anti-myeloma effect of combination therapy with BT062 and Lenalidomide Female mice were inoculated subcutaneously with MOLP 8 human myeloma cells. Treatment with BT062 alone or in combination with Lenalidomide was started on day 11 after tumor inoculation. BT062 is used in concentrations of 100 pg, 200 g and 400 g alone and in combination with Lenalidomide which is dosed in an intraperitoneal form at 100 mg / kg on days 1 to 5 and days 8 to 12. An animal control group received Saline buffered with phosphate (PBS) using the same program y. administration route .. Tumor growth was monitored when measuring the tumor size and calculated with the formula of length x width x height x 1/2, determined on days 10, 14, 18 and 21.

Synergy is calculated as follows (Yu et al., 2001; Gunaratnam et al., 2009): PROPORTION (r) = Expected FTV (combination) / FTV observed (combination) FTV: Fractional tumor volume = average tumor volume (test) / average tumor volume (control) A relationship A > 1 is considered synergistic, while r < 1 is less than additive.

The relation (r) is, when it is greater than 1, referred to here as "SYNERGY RELATIONSHIP".

As can be seen in Table 15, the synergy was observed after 28 days in BT062 concentrations of 200 ig and 400 ug.

BT062 100 + Len Days BT062 100 Lenalidomlda (observed) 10 0.93 1.00 0.97 14 0.75 0.82 0.59 17 0.52 0.45 0.23 21 0.53 0.42 0.19 24 0.44 0.55 0.18 28 0.33 0.46 0.17 BT062 200 + Len BT062 200 Lenalidomide (observed) 10 1.02 1.00 1.00 14 0.45 0.82 0.51 17 0.13 0.45 0.14 21 0.08 0.42 0.07 24 0.11 0.55 0.06 28 0.13 0.46 0.03 BT062 400 + Len BT062 400 Lenalidomlda (observed) 10 0.94 1.00 0.91 14 0.44 0.82 0.24 17 0.09 0.45 0.06 21 0.04 0.42 0.04 24 0.04 0.55 0.03 28 0.04 0.46 0.01 Cont.

Proportion Days BT062 100 + Len expected (exp / obs) 10 0.93 0.96 14 0.61 1.04 17 0.23 1.02 21 0.22 1.19 24 0.24 1.30 28 0.15 0.90 Proportion BT062 100 + Expected len (exp / obs) 10 1.02 1.02 14 0.37 0.73 17 0.06 0.41 21 0.03 0.45 24 0.06 1.08 28 0.06 1.86 Proportion ?? 062 100 + Len expected (exp / obs) 10 ¾ 0.95 1.04 14 0.36 1.49 17 0.04 0.63 21 0.02 0.44 24 0.02 0.80 28 0.02 1.43 Table 16: Volume of fractional tumor in OLP xenografts 8.

Different concentrations of BT062 either alone or in combination with Lenalidomide have been administered in tumor-containing xenograft. FTV represents the relative tumor volume. Synergistic effects are determined using expected FTV ratio values against observed FTV. A relationship > 1 indicates synergy.

Dosage per T / C (%) Injection agent Total dose (DAY 17) PBS (0.2 ml) - - BT062 100 ug / kg 100 ug / kg 35 BT062 200 ug / kg 200 ug / kg 14 BT062 400 ug / kg 400 ug / kg 9 Lenalidomide 100 mg / kg lg / kg 31 BT062 100 ug / kg 100 ug / kg 19 Lenalidomide 100 mg / kg lg / kg BT062 200 ug / kg 200 ug / kg 12 Lenalidomide 100 mg / kg lg / kg BT062 400 ug / kg 400 ug / kg 6 Lenalidomide 100 mg / kg lg / kg Cont.

Surviving Regressions free tumor day Full Partial Agent 77 Result PBS 0/6 0/6 0/6 BT062 0/6 0/6 0/6 Active BT062 0/6 0/6 0/6 Active BT062 4/6 1/6 0/6 Highly active Lenalid 0/6 0/6 0/6 Active measured BT062 0/6 0/6 0/6 Activa Lenalido measure BT062 2/6 0/6 0/6 Active lenalido measure BT062 5/6 4/6 0/6 Highly active Lenalido measure Table 17: Combination of Lenalidomide BT062: effects at different doses.

Figure 28 shows the effect of combination therapy on mean tumor volume (TV) in a mouse-xenograft model. The result shows additive effects of the combination. Please refer to Table 16 for the synergy relationship.

Example 2 Anti-myeloma effect of combination therapy with BT062 and VELCADE VELCADE has been evaluated as a potential multiple myeloma drug combination partner for BT062 in Xenograft studies using OLP8 multiple myeloma cells (IMGN Inc.). Treatment with BT062 alone or in combination with VELCADE was started 11 days after tumor implantation. BT062 was used in concentrations of 100 μg, 200 pg and 400 ig alone and in combination with VELCADE that was dosed at 1 mg / kg on days 1, 4, 8 and 11. An animal control group received phosphate buffered saline ( PBS) using the same program and administration route. Tumor growth was monitored by measuring the tumor size and calculated with the formula length x height x width x 1/2, determined on days 10, 14, 17, 21, 24 and 28, respectively Synergy was calculated as in Example 1 of the combination studies.

As can be seen in Table 18, synergy is observed in the BT062 combination with VELCADE on day 25 in all BT062 dose regimens. R values reported in the literature are even higher (Yu et al., 2001).

BT062 100 + Velcade Day BT062 100 Velcade (observed) 10 1.06 1.05 1.04 14 0.74 0.84 0.56 18 0.44 0.96 0.28 21 0.39 0.80 0.23 25 0.48 0.95 0.26 BT062 200 + Vel Days BT062 200 Velcade (observed) 10 1.02 1.05 1.07 14 0.52 0.84 0.45 18 0.13 0.96 0.10 21 0.10 0.80 0.05 25 0.10 0.95 0.04 BT062 400 + Vel Days BT062 400 Velcade (observed) 10 1.09 1.05 1.04 14 0.45 0.84 0.43 18 0.08 0.96 0.09 21 0.05 0. 80 0.04 25 0.04 0.95 0.02 Cont. proportion Expected day (exp / obs) 10 1.12 1.07 14 0.62 1.11 18 0.42 1.54 21 0.31 1.38 25 0.46 1.75 proportion Expected days (exp / obs) 10 1.12 1.07 14 0.44 0.98 18 0.12 1.19 21 0.08 1.47 25 0.09 2.09 Relation of Days expected synergy (exp / obs) 10 1.15 1.10 14 0.38 0.88 18 0.08 0.89 21 0.04 0.98 25 0.03 1.36 Table 18: Combination treatment with VELCADE.

Fractional tumor volume (FTV) represents the average tumor volume (test) / average relative tumor volume (control). The expected FTV ratio (combination) against observed FTV (observed). Ratio value > 1 indicates synergy, values less than 1 indicate an additive effect.

VELCADE combination Days of treatment (TX date Starting dose (T- Exterpor = day 10 C) inocpost minium T / C in cells Inoc agent ) (%) log days PBS (0.2 mi) Day 1 - - - BT062 100 Day 1 43 5.5 0.5 ug / kg BT062 200 Day 1 11 14.5 1.3 ug / kg BT062 400 Day 1 7 31.5 2.8 ug / kg Velcade 1 mg / kg days 1, 100 0.5 0.0 4, 8, 11 BT062 100 Day 1 20 10.5 0.9 ug / kg Velcade 1 mg / kg days 1, 4, 8, 11 BT062 200 Day 7 23.5 2.1 ug / kg Velcade 1 mg / kg days 1, 4, 8, 11 BT062 400 Day 1 7 36.5 3.2 ug / kg Regressions Free survivors Complement tumor day Resul¬ Partial Agent to 67 PBS 0/6 0/6 0/6 BT062 0/6 0/6 0/6 Inacti vo BT062 1/6 0/6 0/6 Active BT062 4/6 2/6 0/6 Highly active Velcade 0/6 0/6 0/6 Inacti o BT062 1/6 0/6 0/6 Active Velcade BT062 4/6 1/6 0/6 Alta¬ Velcade mind active BT062 6/6 0/6 0/6 Highly active Table 19: VELCADE combination BT062: effects at different doses.

The. Figure 29 shows the effect of .la. combination therapy in mean tumor volume (TV) in a mouse xenograft model. The result shows that in the model used, treatment with VELCADE alone had no effect on tumor volume. The combination with BT062 provides synergistic effects. Please refer to Table 18 for the synergy relationship.

Example 3: BT062 / Melphalan ^ RPMI cells have been implanted subcutaneously in nude mice. Mice were randomized when the tumor reached a total volume of approximately 100 mm3. BT062 was injected intravenously at 2 different concentrations: 400 pg / kg and 100 g / kg; each based on the molecular weight of bound DM4. PBS served as a negative control. By group, 8 mice with one tumor each (unilateral implant) were used. BT062 was dosed weekly followed by Melfalan once a week (3 mg / kg) one day after intraperitoneal injection of BT062.

The results are shown in Figure 30.

Once given the above description, many other characteristics, 'modifications' "and improvements will be apparent to the person with dexterity.These other characteristics, modifications and improvements are therefore considered as part of this invention, the scope of which will be to be determined by the compendium of the invention and the following claims.

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Claims (55)

1. A method for treating a disease associated with CD138-expressing target cells, characterized in that it comprises: administering to a patient in need thereof, an effective amount of an immunoconjugate comprising at least one targeted agent that targets or targets cells expressing CD138, and at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, and wherein the effective amount is a tolerable amount.

2. The method according to claim 1, characterized in that the immunoconjugate is administered to the subject in an amount of 5 mg / m2 to 200 mg / m2 or pharmacokinetic equivalent of 5 mg / m2 to 200 mg / m2 when administered in combination with a agent to treat adverse side effects.

3. The method according to claim 1 or 2, characterized in that a maximum concentration of the immunoconjugate in the plasma of the subject between 0 to 2 hours after the end of a first administration is less than 50%, preferably less than 40%, more preferably less than 30%, even more preferable less than 20%, or even less than 10% of a theoretical maximum concentration for the immunoconjugate.

4. The method according to claim 3, characterized in that the immunoconjugate is administered at least four times and a maximum concentration of the immunoconjugate in the plasma of the subject between 0 to 2 hours after the end of each of the administrations is less than 55%, preferably less than 50%, more preferably less than 40%, still more preferable less than 30%, less than 20% or even less than 10% of the theoretical maximum concentration for the immunoconjugate.

5. The method in accordance with the claim 1, characterized in that the immunoconjugate is administered in a dose, preferably a single repeated dose, not greater than about 10, 20, 30, 40, 80, 90, 100 or 120 mg / m2, an average daily dose of about 400] ig / m2 to approximately 6 mg / m2, including approximately 500 μg / m2, approximately 1 mg / m2, approximately 2 mg / m2, approximately 3 mg / m2, approximately 4 mg / m2, approximately 5 mg / m2, and / or an average weekly dose of about 3 mg / m2 to about 40 mg / m2, including about 5 mg / m2, about 10 mg / m2, about 15 mg / m2, about 20 mg / m2, about 25 mg / m2, about 30 mg / m2 or approximately 35 mg / m2.

6. The method according to any of the preceding claims, characterized in that by approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 210 or more days is maintained the stable disease.

7. The method according to any of the preceding claims, characterized in that for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 treatment cycles each of approximately 3 weeks remains disease stable the disease.

8. The method according to claim 7, characterized in that at least stable disease is maintained by 5, 6, 7, 8, 9 or 10 treatment cycles of 20 mg / m2.

9. The method according to claim 8, characterized in that a minor response is observed after 8 treatment cycles.

10. The method according to any of the preceding claims, characterized in that the method results in a stable disease, a response, in particular a minor response, a partial response, a very good partial response, a severe complete response or a durable complete response by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 treatment cycles or more wherein the treatment cycles each comprise approximately 3 weeks with an administration of the immunoconjugate on day 1 of each treatment cycle or by 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77 or 82 days, respectively.

11. The method according to any of the preceding claims, characterized in that the immunoconjugate is administered to the subject in an amount of 5 mg / m2 or 10 mg / m2 to less than 160 mg / m2, preferably 150 mg / m2, 140 mg / m2, 130 mg / m2 or 120 mg / m2.

12. The method according to claim 3, characterized in that the maximum concentration is less than 3 pg / ml per 10 mg / m2, less than 8 μg / ml per 20 mg / m2, lower by 15 μg / ml per 40 mg / m2 , less than 25 μg / ml per 80 mg / m2, less than 30 ug / ml per 120 mg / m2.

13. The method according to claim 4, characterized in that the maximum concentration is less than 14 μg / ml for 20 mg / m2, less than 15 μg / ml for 40 mg / m2 or less than 25 μg / ml for 80 mg / m2 .

14. The method according to claim 1, characterized in that the CD138 in the patient is expressed in the target cells and in non-target cells, wherein the administration results in elimination of the moderate or slow immunoconjugate in plasma, and in which the cells do not target that express CD138, in particular epithelial cells, are not substantially affected.

15. The method according to claim 14, characterized in that the level of expression of CD138 in the target and non-target cells expressing CD138 is comparable.

16. The method according to claim 14 or 15, characterized in that the effective amount administered is less than 200 mg / m2 or less than a pharmacokinetic equivalent of 200 mg / m2 when administered in combination with an agent to treat adverse side effects and in wherein the administration results in a response in the subject, preferably after less than 40, 30, 20, 15, 10, 9, 8, 7, 6, 5 hours.

17. The method according to claim 16, characterized in that the effective amount is greater than 120 mg / m2.

18. The method according to claim 14 or any of the following claims, characterized in that the effective amount is administered as a single dose, a single dose repeated or in multiple doses.

19. The method according to claim 18, characterized in that a cmax value after each administration is greater than 55% of a theoretical cmax value.

20. The method according to claim 14, or any of the following claims, characterized in that the administration results in M or FLC protein levels of at least stable disease, a minor response or a partial response in the subject, preferably after a first administration.

21. The method according to any of the preceding claims, characterized in that the immunoconjugate comprises an antigen binding region (ABR) against CD138, and an additional antibody region, wherein at least part of the additional antibody region is an antibody and confer the properties of isotype IgG4.

22. The method according to claim 21, characterized in that the immunoconjugate comprises nBT062 or a targeted antibody having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with nBT062 or corresponds to BT062.

23. The method according to any of the preceding claims, characterized in that it consists essentially of administering a pharmaceutical composition comprising the immunoconjugate and an acceptable pharmaceutical carrier, wherein an active ingredient of the composition consists essentially of the immunoconjugate.

24. The method according to any of the preceding claims, characterized in that the immunoconjugate is administered intravenously.

25. The method according to claim 24, characterized in that the immunoconjugate is administered intravenously in a single repeated dose.

26. The method according to any of the preceding claims, characterized in that the disease is a plasmaproliferative disorder associated with the expression CD-138, such as multiple myeloma, in particular relapsed or refractory multiple myeloma.

27. The method according to claim 1 or 2, characterized in that the disease expressing CD138 in target cells is selected from the group consisting of renal cell carcinoma, endometrial cancer,. cervical cancer, prostate adenocarcinoma, pancreatic carcinoma, gastric cancer, bladder cancer, mammary carcinoma, hepato-carcinoma, colorectal carcinoma, colon carcinoma, squamous cell carcinoma, lung cancer in particular squamous cell lung carcinoma, non-Hodgkin's lymphoma, Thymus, uterus, urinary or ovarian carcinoma.

28. The method according to any of the preceding claims, characterized in that the disease is associated with bone pain and / or bone complications and wherein the administration of the immunoconjugate reduces bone pain and / or - - bone complications, preferably at an acceptable level.

29. The method according to claim 1, characterized in that the immunoconjugate exceeds a refractory phenotype. ^

30. Method for treating a disease associated with CD138-expressing target cells, characterized in that it comprises (i) identifying a disease, associated with target cells expressing CD138, such as multiple myeloma, and not responding or responding poorly to treatment with one or more agents cytotoxic agents including immunomodulators and / or proteasome inhibitors, and (ii) administering to the subject, preferably intravenously, an effective amount of an immunoconjugate of claim 2, wherein the subject does not respond or responds poorly to treatment with one or more cytotoxic agents including immunomodulators and / or proteasome inhibitors, and wherein the disease is treated.

31. A method for treating a disease associated with CD138 expressing target cells, characterized in that it comprises administering to a patient requiring this treatment and exhibiting high levels of SCD138, such as more than 50 ng / ml, more than 60 ng / ml, more than 70 ng / ml more than 80 ng / ml, more than 100 ng / ml, more than 150 ng / ml, more than 200 ng / ml, more than 200, 400, 500, 600, 700, 800, 900, - - 1000, 1100, 1200, 1300, 1400, 1500 ng / ml, an effective amount of the immunoconjugate of claim 1, wherein an amount as low as 20 mg / m2 or as low as 40 mg / m2 is effective to provide a response such as a minor response.

32. The method according to claim 31, characterized in that the response results from the selective binding of the immunoconjugate.

33. The method according to claim 31, characterized in that the subject does not respond or responds poorly to treatment with cytotoxic agents including immunomodulators such as inhibitors of lenalidomide and / or proteasome such as bortezomib.

34. An anti-cancer combination characterized in that it comprises: at least one cytotoxic agent and at least one immunoconjugate comprising a targeted agent that targets cells expressing CD138, and at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, wherein (a) the combination has a synergy ratio greater than 1, greater than 1.1, greater than 1.2, greater than 1.3, greater than 1-4, or (b) the combination has a Logio cell death greater than 2, or (c) the combination has a synergistic ratio of about 1 and the effector molecule and the cytotoxic agent have modes of interference action, and wherein the anti-cancer combination is a pharmaceutical composition or a device comprising separate containers of the cytotoxic agent at least and the immunoconjugate at least.

35. The anti-cancer combination according to claim 34, characterized in that the cytotoxic agent is a proteasome inhibitor, an immunomodulatory agent or an anti-angiogenic agent, a DNA alkylating agent or a mixture of two or more thereof.

36. The anti-cancer combination according to claim 34, characterized in that the cytotoxic agent is bortezomib, thalidomide, lenalidomide, melphalan or a mixture of two or more thereof.

37. The anti-cancer combination according to any of claims 34 to 36, characterized in that the targeted agent is an engineered target antibody in cells expressing CD138.

38. The anti-cancer combination according to claim 37, characterized in that the engineered antibody comprises an ABR against CD138, and an additional antibody region in, where at least part of the additional antibody region is from a human antibody and confers properties of isotype IgG4.

39. The anti-cancer combination according to claim 38, characterized in that the immunoconjugate comprises nBT062 or a targeted antibody having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity with nBT062 or corresponds to BT062 .

40. The anticancer combination according to any of claims 33 to 38, characterized in that the effector molecule and the cytotoxic agent have modes of interference action, and wherein these modes of action preferably involve microtubule inhibition or cell cycle brake induction. .

41. The anti-cancer combination according to claim 40, characterized in that the interference action mode is cell cycle brake induction | and wherein the cytotoxic agent is melphalan, bortezomib and lenalidomide or thalidomide.

42. The anti-cancer combination according to any of claims 34 to 39, characterized in that the effector molecule and the cytotoxic agent have modes of action without interference.

43. The anti-cancer combination according to any of claims 34 to 42, characterized in that the anti-cancer combination is a pharmaceutical composition comprising at least one acceptable pharmaceutical excipient.

44. The anti-cancer combination according to any of claims 34 to 42, characterized in that the anticancer combination is an equipment comprising in a container the immunoconjugate, in a second container the cytotoxic agent at least and instructions on how to use the components of the equipment.

45. A method for treating a disease associated with CD138-expressing target cells, characterized in that it comprises: administering to a patient in need thereof, an effective amount of the anti-cancer combination according to any of claims 34 to 44 or an anti-cancer combination comprising at least one cytotoxic agent and at least one immunoconjugate comprising a targeting agent in cells expressing CD138 and at least one effector molecule, wherein the targeted agent is functionally linked to the effector molecule to form the immunoconjugate, and wherein the immunoconjugate overcomes a refractory phenotype. )

46. A method for treating a non-proliferative plasma disease associated with CD138-expressing target cells, characterized in that it comprises: administering to a subject that requires it or cells of the non-proliferative plasma disease, an effective amount of an immunoconjugate comprising at least one targeting agent in cells expressing CD138, and at least one effector molecule, wherein the targeting agent is unionally linked to the effector molecule to form the immunoconjugate, wherein CD138 in the subject, is expressed in the target cells and in non-target cells at comparable levels or where CD138 in the subject is expressed in the target cells at levels lower than those of non-target cells expressing CD138.

47. The method according to claim 46, characterized in that the non-target cells expressing CD138 are epithelial cells.

48. The method according to claim 45, characterized in that the target cells of the disease shed CD138 over a period of 24 hours, 2, 3, 4, 5, 6 days.

49. The method according to claim 48, characterized in that the disease is mammary carcinoma.

50. The method according to claim 45 or 46, characterized in that the immunoconjugate induces remission of a solid tumor.

51. The method according to claim 50, characterized in that the solid tumor is a pancreatic carcinoma or mammary carcinoma.

52. The method according to claim 50 or 51, characterized in that the remission is followed by a time interval that is free of regrowth of the tumor.

53. The method according to claim 45 or 46, characterized in that the disease is renal cell carcinoma, endometrial cancer, cervical cancer, prostate adenocarcinoma, pancreatic carcinoma, gastric cancer, bladder cancer, mammary carcinoma, hepato-carcinoma, colorectal carcinoma , colon carcinoma, squamous cell carcinoma, lung cancer, in particular squamous cell lung carcinoma, non-Hodgkin's lymphoma, thymus, uterus, urinary or ovarian carcinoma.

54. The method according to claim 50, characterized in that the tumor is a mammary carcinoma, which is a negative estrogen receptor and / or a negative progesterone receptor and / or a herceptin resistant.

55. The method according to any of the preceding claims, characterized in that the targeted antibody is an engineered antibody.